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Contents

1.0 Introduction

2.0 Discussion

• 2.1 How do NSAIDS suppress prostaglandins?

• 2.2 NSAIDs and healing: What are the research findings?

  • The role of prostaglandin E2 (PGE2) receptor signalling in wound healing

  • Do NSAIDs mimic accelerated ageing?

3.0 Parting shots

• 3.1 Supply chain impacts on statins and aspirin

• 3.2 Caught red-handed: Wes deleting Epstein-related posts

4.0 Concluding remarks

5.0 Disclaimer

6.0 References

1.0 Introduction

Many of us take NSAIDs each day, especially low-dose (75mg) enteric-coated aspirin. In simple terms, it acts as an anti-inflammatory agent, as part of its painkilling mechanisms. Low-level inflammation is one of the leading underlying causes of many diseases, including cancer and cardiovascular disease (CVD).1 Multiple studies have demonstrated a consistent preventative effect on the risk of us dying from cancer, in the order of 20-30%.2

As for CVD, 8,167 predominantly Aboriginal adults were included and followed in a study from June 2009 to June 2017. Of these, 1,865 were regular aspirin users. 6,302 were non-users, with a mean follow-up of 4 years, and an average hospitalisation rate of 6.4 per person. The study indicated a reduction in the rate of ischaemic heart disease (IHD) in the aspirin-takers of 16% vs non-users. The findings for deaths attributed to CVD were reduced by 56%.

Although not considered statistically significant due to the small number of events during the study, these findings became statistically significant at 67% when adjusted for confounders. Although higher than previous studies, this was thought to be due to the younger age of the Aboriginal participants (mean age 42, vs 55-71 in other studies), as this group is particularly prone to earlier onset of multiple cardiovascular risk factors, such as diabetes, hypertension, dyslipidaemia, albuminuria, obesity and renal disease. The preventative effects of aspirin thus became all the more significant.3

What is an NSAID?

Aspirin is an example of a non-steroidal anti-inflammatory drug.

In contrast, the many drawbacks of steroid drugs were discussed here:

NSAID medications are valued because of their ability to reduce pain, decrease fever, and lower inflammation. They almost need no introduction, as most of us take them regularly to treat headaches, menstrual cramps, strains, stiffness and joint pain caused by arthritis or injury.

They work by inhibiting cyclo-oxygenase (COX) enzymes to block prostaglandin synthesis.

The two most significant COX enzymes are COX-1 and COX-2. They convert a long-chain omega-6 polyunsaturated acid, arachidonic acid (ARA), into prostaglandins.

Although all NSAIDs are COX inhibitors, not all COX inhibitors are NSAIDs. For instance, acetaminophen is a selective COX-2 inhibitor.

ARA is found almost exclusively in animal-based foods, such as meat, eggs, and dairy.

In case you were wondering, sorry Bill, but most lab-grown meats only have half the polyunsaturated fatty acids (PUFAs) and twice the less healthy monounsaturated fatty acids (MUFAs) of natural cells.4

Apart from eating Bill’s robo-trash junk, we consume most of our MUFAs from plant sources, including olive oil, avocados, canola oil, high-oleic sunflower oil, macadamia nuts, hazelnuts, almonds, and cashews. These aren’t necessarily bad sources, but they would leave you deficient in animal-sourced AHA if that’s all you lived off.

The reason for writing this Substack is a hypothesis which links the drawbacks of eating lab-grown meat with the potential drawbacks of regularly taking NSAIDs.

As with my deep-dive into statins, nature doesn’t like to waste anything, and we didn’t evolve these long chemical synthesis pathways for nothing. They are often quite expensive, in terms of energy and molecular input. There is likely to be a survival aspect to many of these pathways, and blocking them with synthetic drugs may have unexpected, negative consequences.

In short, what may be some of the secondary effects of inhibiting prostaglandin production? Could NSAIDs slow down the repair of injuries, including fractures, torn ligaments and tendons?

If you have a long-standing injury that doesn’t seem to be healing properly, or you have frequent setbacks, then this Substack may be of particular interest to you.

Correlation isn’t causation, but as with the multi-hit theory of cancer, the status of our health reflects the sum total of many factors of influence, however minor in importance they may appear to be.

Emphasis is mine in bold, and some passages are lightly reformatted for legibility on portable devices.

2.0 Discussion

2.1 How do NSAIDS suppress prostaglandins?

As both a benefit and a potential drawback, even low doses of aspirin irreversibly inhibit both COX-1 and COX-2 by permanently acetylating these enzymes. With COX-2, it happens at the site of a critical serine residue called Ser516. Aspirin also acts as an antiplatelet agent by mediating the reduction of a clotting agent called thromboxane A₂ (TXA₂), which is great for helping to prevent strokes and thrombosis.5

Acetylation of an enzyme is a post-translational modification (PTM). An acetyl group (CH₃CO) is transferred from acetyl-CoA to specific amino acid residues, typically lysine, by enzyme acetyltransferases (KATs). PTM changes the residue’s charge from positive to neutral. This can effectively act as a molecular switch by changing the protein’s conformation, function, stability, and localisation. Most enzymes are proteins.6

“Prostaglandins (PGs) are lipid mediators, generated from arachidonic acid (AA) metabolism via cyclooxygenases (COX). They were discovered in the 1930s as regulators of blood pressure and smooth muscle contraction [1]. The distribution of synthases and receptors for each PG is different in various cell types, and PGs are activated via either paracrine or autocrine signaling on the surface of each cell type [2]. PGs bridge the interactions between various immune-modulating cells, and are considered key players in regulating pro-inflammatory and anti-inflammatory responses [3].”7

It can take as long as 10 days for your platelet levels to recover after you stop taking platelet inhibitors. 4 days is typical for aspirin.

Clopidogrel is an antiplatelet agent, but it’s not a COX-2 inhibitor.

Reversal of the anti‐platelet effects of aspirin and clopidogrel (2012)

Background:

Guidelines recommend stopping aspirin and clopidogrel 7 to 10 days before surgery to allow time for replacement of permanently inhibited platelets by newly released uninhibited platelets.

Objectives:

The purpose of the present study was to determine the rate of offset of the anti‐platelet effects of aspirin and clopidogrel after stopping treatment and the proportion of untreated donor platelets that are required to reverse their anti‐platelet effects.

Methods:

Cohort 1 consisted of 15 healthy subjects who received aspirin 81 mg day⁻¹ or clopidogrel 75 mg day⁻¹ for 7 days and underwent serial blood sampling until platelet function testing results normalized. Cohort 2 consisted of 36 healthy subjects who received aspirin 325 mg day⁻¹, clopidogrel 75 mg day⁻¹, aspirin 81 mg day⁻¹ plus clopidogrel 75 mg day⁻¹ or no treatment for 7 days and underwent a single blood sampling.

Results:

In cohort 1, arachidonic acid (AA)‐induced light transmission aggregation (LTA) returned to baseline levels in all subjects within 4 days of stopping aspirin, coinciding with the partial recovery of plasma thromboxane B₂ concentrations.

ADP‐induced LTA did not return to baseline levels until 10 days after stopping clopidogrel. In cohort 2, AA‐induced LTA in patient treated with aspirin reached control levels after mixing with 30% untreated donor platelets whereas ADP‐induced LTA in patients treated with clopidogrel reached control levels only after the addition of 90% or more donor platelets.

Conclusions:

Platelet aggregation recovers within 4 days of stopping aspirin but clopidogrel must be stopped for 10 days to achieve a normal aggregatory response.

More: https://www.sciencedirect.com/science/article/pii/S1538783622062250

2.2 NSAIDs and healing: What are the research findings?

Evidence-based medicine (EBM) has got a bad press in some circles because of its misuse, but the underlying principles are sound:

“Evidence-based medicine de-emphasizes intuition, unsystematic clinical experience, and pathophysiologic rationale as sufficient grounds for clinical decision making and stresses the examination of evidence from clinical research.”8

We can look for evidence of harm in several stages. These may include studies which are:

• In silico. Computer modelling or simulations may be used to study biochemical mechanisms, for example, to model protein structure, function and behaviour.

• In vitro. Experiments are conducted in a test tube, culture dish, or elsewhere outside a living organism.

• In vivo. Biological processes are studied in a living organism.

• Clinical. A research project involving human volunteers which is designed to evaluate the safety, efficacy, and mechanisms of new drugs, medical devices, diagnostic tools, or behavioural interventions.

Where large sums of money are involved, especially for Big Pharma, you inevitably risk research being compromised or suppressed if that research may lead to reduced sales of that drug. And the NSAID market is huge:

Non-steroidal Anti-inflammatory Drugs Market to Reach USD 36.10 Billion by 2030 – New Research Explores Key Dynamics

Research and Markets

23 January 2024

The comprehensive analysis of the Non-steroidal Anti-inflammatory Drugs Market provides an in-depth look into the industry's future projections, with a spotlight on high-growth areas such as arthritis medications and regional trends within the Asia-Pacific region. Projections estimate the market, valued at USD 21.71 billion in 2023, to grow at a steady CAGR of 7.53%, achieving USD 36.10 billion by the year 2030.

More: https://uk.finance.yahoo.com/news/non-steroidal-anti-inflammatory-drugs-032300264.html

Yes, the Willie Sutton “why do you rob banks?” law very much applies to NSAIDS.

Although some specific NHS websites warn that NSAIDs may impair healing, it isn’t widely advertised, and you won’t see any warnings on the package insert. For instance, there is no such warning on the web page that most of us are likely to read:

Mission accomplished.

This is a package insert for low-dose aspirin. None of the possible secondary effects caused by inhibiting prostaglandins gets a mention, such as if you have a fracture or tissue injury that needs time to heal:

Aspirin Low Dose Enteric Coated: Package Insert / Prescribing Info

Package insert / product label
Dosage form: tablet, coated
Drug classes: Platelet aggregation inhibitors, Salicylates

Medically reviewed by Drugs.com. Last updated on Sep 24, 2025.

Active Ingredient in Each tablet:

Aspirin 81 mg (NSAID) Non-steroidal anti-inflammatory drug.

Ask a doctor before use if: stomach bleeding warning applies to you, you have a history of stomach problems, such as heartburn, you have high blood pressure, heart disease, liver cirrhosis, or kidney disease; you are taking a diuretic, you have asthma, you have not been drinking fluids, you have lost a lot of fluid due to vomiting or diarrhea

Uses: temporarily relieves minor aches and pains: for other uses, see your doctor, but do not use for more than 10 days without consulting your doctor because serious side effects may occur.

Reye’s syndrome: Children and teenagers who have or are recovering from chicken pox or flu-like symptoms should not use this product. When using this product, if changes in behavior with nausea and vomiting occur, consult a doctor because these symptoms could be an early sign of Reye’s syndrome, a rare but serious illness.

Allergy alert: Aspirin may cause a severe allergic reaction which may include: hives, facial swelling, shock, asthma (wheezing).

Stomach bleeding warning: This product contains an NSAID which may cause severe stomach bleeding. The chance is higher if you: are age 60 or older, have had stomach ulcers or bleeding problems, take a blood thinning (anticoagulant) or steroid drug, take other drugs containing prescription or non prescription NSAIDs (aspirin, ibuprofen, naproxen, or others), have 3 or more alcoholic drinks every day while using this product, take more or for a longer time than directed.

Ask a doctor or pharmacist before use if you are taking a prescription drug for diabetes, gout, or arthritis; under a doctor’s care for any serious condition; taking any other drug.

Do not use if you are allergic to aspirin or any other pain reliever/fever reducer.

Stop use and ask a doctor if: you experience any of the following signs of stomach bleeding: feel faint, have bloody or black stools, vomit blood, have stomach pain that does not get better, pain gets worse or lasts more than 10 days, fever gets worse or lasts more than 3 days, redness or swelling is present in the painful area, any new symptoms appear.

If pregnant or breast-feeding, ask a health profession before use. It is especially important not to use aspirin during the last 3 months of pregnancy unless definitely directed to do so by a doctor because it may cause problems in the unborn child or complications during delivery.

Keep out of the reach of children. In case of overdose, get medical help or contact a Poison Control Center right away.

Uses - temporarily relieves minor aches and pains for other uses, see your doctor, but do not use for more than 10 days without consulting your doctor because serious side effects may occur.

More: https://www.drugs.com/pro/aspirin-low-dose-enteric-coated.html

The only clue that there may be secondary mechanisms of harm (pathologies) is that you are advised to consult your doctor before taking it for more than 10 days, because unspecified “serious side effects may occur”.

From 2021, Stephan A. Schug asked the question and discussed the evidence.

Note that he declared a significant conflict of interest (COI).

Do NSAIDs Really Interfere with Healing after Surgery?

Abstract

Perioperative analgesia should be multimodal to improve pain relief, reduce opioid use and thereby adverse effects impairing recovery. Non-steroidal anti-inflammatory drugs (NSAIDs) are an important non-opioid component of this approach.

However, besides potential other adverse effects, there has been a longstanding discussion on the potentially harmful effects of NSAIDs on healing after surgery and trauma.

This review describes current knowledge of the effects of NSAIDs on healing of bones, cartilage, soft tissue, wounds, flaps and enteral anastomoses. Overall, animal data suggest some potentially harmful effects, but are contradictory in most areas studied. Human data are limited and of poor quality; in particular, there are only very few good randomized controlled trials (RCTs), but many cohort studies with potential for significant confounding factors influencing the results.

The limited human data available are not precluding the use of NSAIDs postoperatively, in particular, short-term for less than 2 weeks. However, well-designed and large RCTs are required to permit definitive answers.

A universal component of most multimodal analgesic approaches are non-steroidal anti-inflammatory drugs (NSAIDs). Ideally combined with paracetamol, they provide well-proven beneficial effects with regard to improved analgesia and reduced opioid requirement, as demonstrated in a network meta-analysis [6].

I discussed in the acetaminophen Substack why this sort of narrative leads to accidental overdose:

While paracetamol can be used nearly universally due to negligible adverse effects, NSAIDs carry a much higher risk of adverse effects, limiting their use [7].

The introduction of cyclooxygenase-2 (COX-2) selective NSAIDs (coxibs) has reduced these risks with regard to gastrointestinal ulcers, bleeding complication and bronchospasm [4]. Coxibs may even carry a lower risk than some nonselective NSAIDs (nsNSAIDs) with regard to kidney compromise and a similar risk of cardiovascular events [8]; in a meta-analysis, the injectable coxib parecoxib used perioperatively had no statistically different risk than for all adverse effects [9].

One issue which continues to be discussed in this context is the effect of NSAIDs (nsNSAIDs and coxibs) on postoperative and posttraumatic healing of soft tissues, bones and anastomoses, here with the potential risk of anastomotic leakage.

2. Results

2.1. Bone Healing

Bone tissue undergoes a continuous remodelling process involving ongoing resorption, formation and mineralisation. Prostaglandins, in particular prostaglandin E2 (PGE2), are involved in maintaining this process and essential to the balance of osteoblasts and osteoclasts [10].

Bone healing after a fracture is different from healing of other injuries, in so far as it is a process of regeneration of normal bone tissue and not formation of a scar. It is therefore the result of the normal remodelling process and thereby dependent on prostaglandins [11].

This has resulted in theoretical concerns about the effects of NSAIDs on bone healing, as their main effect is inhibition of prostaglandin synthesis. These concerns may in particular relate to coxibs, as COX-2 is the dominant cyclo-oxygenase in osteoblasts.

Support for the hypothesis is from in vitro, in vivo, and some clinical studies:

In line with these physiologically founded concerns, an initial study of indomethacin use after closed femoral fracture in a rat model showed delays in fracture healing and formation of inferior callus predisposing to non-union [12].

This triggered a discussion on the potentially harmful effects of NSAIDs on bone healing, which is still ongoing. A number of subsequent animal studies, mainly in rodents, confirmed the detrimental effect of NSAIDs, in particular coxibs, on early fracture repair and the subsequent bone density and strength [13]. However, these effects are not consistently reported and translation to demonstrable effects on fracture healing in clinical settings in human patients is even less convincing.

NSAIDs may double your risk of delayed or failed bone union, but not if taken for 2 weeks or less.

A confounding factor may be that you are taking the NSAID because of the pain caused by a poorly healing fracture. I would counter this by saying that the rats they studied didn’t have this option.

A most recent meta-analysis of effects of NSAIDs in humans shows an association between NSAID exposure and delayed union or non-union (OR 2.07; 95% CI 1.19 to 3.61) [14].

However, the authors note the lack of randomised controlled trials (RCTs) and an increased odds ratio in studies of low quality. Confounding factors in the observational cohort studies such as patients using more NSAIDs for longer due to increased pain for a poorly healing fracture could have contributed to these results.

Furthermore, there was no association shown with low dose or short-term use (<2 weeks) (OR 1.68; 95% CI 0.63 to 4.46) or in paediatric patients (OR 0.58; 95% CI 0.27 to 1.21).

2.2. Cartilage Repair

Similar to the concerns about bone healing, there has been an ongoing discussion on the effects of NSAIDs on chondrocytes. In vitro and animal studies report early negative effects on chondrocyte proliferation, but not at later time points, and on chondrocyte differentiation, but again these data are not consistent [26]. This systematic review claims to have identified only one human study; however, this is a study of anterior cruciate ligament repair and will be discussed below [27].

Overall, the conclusions are that it is currently unclear, due to a lack of any studies in humans, what effects NSAIDs have on the outcome of cartilage repair procedure.

2.3. Musculoskeletal Soft Tissue Healing (Tendons and Ligaments)

A systematic review of animal studies on tendon-to-bone healing was compromised by heterogeneous outcome reporting [28]; a subgroup analysis of homogenous animal studies showed no effect by NSAIDs.

Rabbit rotator cuff (RC) pathology is investigated to simulate human tendon tears, atrophy, and healing. A lower load before failing under tension implies that the drug negatively impacts the healing process, resulting in a weaker tendon-to-bone attachment (enthesis) and a higher risk of re-tear.

However, in a rabbit model of rotator cuff repair, nsNSAIDs had no (ibuprofen) or only minimal early (flurbiprofen) effects on healing, while coxibs (celecoxib) resulted in lower failure loads at any time point between 3 and 12 weeks [29].

Findings were repeated in a clinical setting in one study, but not in 3 other observational studies; nevertheless, there is insufficient human data to disprove causality.

Similar results were found in a human RCT (n = 180) with higher re-tear rates after use of celecoxib versus ibuprofen and tramadol [30].

The above study was also the only RCT included in a meta-analysis of studies which looked at effects of NSAIDs on soft tissue healing in general [31]. It analysed another 3 observational studies, of which the largest contributed 93% of cases (anterior cruciate ligament repair); it found overall no difference in surgical failure rate and other outcomes between NSAID and no NSAID use (3.6 vs. 3.7%).

There are insufficient human data on the effect of musculoskeletal soft tissue healing, but their use may not have a detrimental effect, possibly with the exception of celecoxib use after rotator cuff repair.

COX-inhibitors (“Coxibs”) may have a negative effect on tissue flap survival and healing, which is also quite concerning and not unexpected:

In an animal study in rats, perioperative celecoxib use had no harmful effects on flap survival and healing compared to non-use [32].

In a retrospective cohort study (n = 138), ketorolac use after head and neck free flaps did not increase bleeding complications or risk of free flap failure [33].

With regard to coxibs, the results are contradictory. An animal study in rats showed a detrimental effect on flap survival with use of parecoxib [34], but not with celecoxib [32].

The only human study here is a cohort study with historical controls with the associated risk of potential confounding factors [35]. The introduction of parecoxib and valdecoxib for two to three weeks postoperatively resulted in an increase of free flap failure from a base line rate of 7% to 29%, which fell to 4% when coxibs were no longer used.

Again, research findings were inconclusive, but one study indicated a doubling of relative risk:

2.5. Anastomotic Leakage

Use of NSAIDs after gastrointestinal surgery is of particular interest, as NSAIDs improve recovery of gastrointestinal function (faster return of flatus, stool passage and oral feeding tolerance) and would therefore enhance postoperative rehabilitation [36].

However, there is an ongoing discussion on the potential of NSAIDs to increase anastomotic leakage. This concern has been partially driven by rodent data showing reduced collagen formation in subcutaneous granulation tissue due to exposure to diclofenac, although anastomotic and skin wound strength were not affected in this study [37].

Two most recent systematic reviews of primarily cohort studies with an overlap of four studies showed an increase in anastomotic leakage rate with all nsNSAIDs; a later smaller one including 8 studies found an OR of 1.79 (95% CI 1.47 to 2.18) [38] and the bigger one including 17 studies an OR of 2.02 (95% CI 1.62 to 2.50) [39].

In the latter meta-analysis, there was no difference if only RCTs were analysed, suggesting a potential risk of bias by basing these systematic reviews to a large extent on cohort studies [39]. It is in particular of note, that in both these systematic reviews, subgroup analysis found no increased risk with selective COX-2 inhibitors.

Overall, healing of gastrointestinal anastomoses is unlikely to be impaired by perioperative use of NSAIDs. This is better documented for coxibs than for nsNSAIDs.

2.6. Wound Healing

Animal studies on wound healing report contradictory results. For example, specifically for coxib use, there are studies which show impaired healing [40], no effect on healing [41] or improved healing [42]. There are no published human data on this issue.

In conclusion, caution is warranted as there is supporting evidence to avoid taking NSAIDs or coxibs for longer than 2 weeks. This is based on theoretical, in vivo and clinical research findings, but more research is needed:

… In the interim, the data currently available should not preclude the use of NSAIDs postoperatively, in particular, when used in the short term for less than 2 weeks. Overall, data suggest that coxibs are safer in the setting of GI anastomoses, but may have more negative effects on bone and soft tissue healing, but again this statement is based on very poor data and may be speculative.

Conflicts of Interest

Stephan Schug was an employee of the University of Western Australia (UWA); within the last 2 years, his employer, and since his retirement, he himself, has received honoraria, consulting fees, and travel support from Andros Pharmaceuticals, Aspen, bioCSL, Grunenthal, Indivior, Janssen, Luye Pharma, Pfizer, Seqirus, and iX Biopharma. None of these payments relate to this manuscript.

More: https://www.mdpi.com/2077-0383/10/11/2359

A review from 2024 by Bennett et al. in the “Wounds Australia Journal” called “The negative impact of medications on wound healing”9 is less opaque in its discussion:

… This review aims to highlight the main types of drugs which negatively impact wound healing. Cancer treatments, non-steroidal anti-inflammatory drugs (NSAIDs), anticoagulants, immunosuppressants, and some antibiotics are all risk factors for cutaneous adverse effects. Identifying drug-induced impaired wound healing is important to counsel patients and their medical practitioners on weighing up the benefits and risks of these medications.

Non-steroidal anti-inflammatory drugs (NSAIDS)

NSAIDs are members of a therapeutic drug class which reduces pain, decreases inflammation, decreases fever, and prevents blood clotting. There are two general types of NSAIDs available: non-selective, and COX-2 selective. COX-2 selective drugs include celecoxib, etoricoxib, and meloxicam.

Most NSAIDs are non-selective and inhibit the activity of both COX-1 and COX-2; examples include naproxen, ibuprofen, diclofenac, indomethacin. These NSAIDs, while reducing inflammation, also inhibit platelet aggregation and increase the risk of gastrointestinal ulcers and bleeds.

Their mode of action is to inhibit inflammatory response and acid mucopolysaccharide synthesis in wounds, inhibit inflammatory mediators derived from arachidonic acid metabolism and platelet aggregation.

NSAIDs inhibit the production of prostaglandins (PGs), which may be the likely mechanism through which NSAIDs impart their deleterious effects on bone healing. By inhibiting the COX enzymes and the subsequent PG production, NSAIDs not only achieve their desired anti-inflammatory effects but also inhibit the increased production of PGs that is necessary for bone healing to occur. Additionally, the inhibition of COX-1 can increase the local ischemia and hypoxia associated with chronic venous ulcers.²⁶

A study found reduced ligament repair and strength in surgically incised medial collateral ligament in 50 rats treated post operatively with a COX-2 inhibitor when compared with the non-treated rats.²⁷

The inhibition of migration and proliferation of tendon cells by NSAIDs has also been demonstrated by Tsai et al.^28,29 In animals treated with indomethacin or parecoxib, the Achilles tendon has decreased tensile strength compared to the control groups.^30,31.

They advise waiting at least 7 days after surgery before taking the NSAID indomethacin.

The authors also refer to in vitro study [32], which suggests that the NSAID indomethacin may have a negative effect on the mitochondrial enzymes in tendon cells.

This makes sense, as tendon cells (tenocytes) rely heavily on mitochondria for energy, and a deficit in these enzymes leads to tendinopathy due to chronic, degenerative changes.10

32. Mallick E, Scutt N, Scutt A, Rolf C. Passage and concentration-dependent effects of Indomethacin on tendon derived cells. Journal of Orthopaedic Surgery and Research. J Orthop Surg Res. 2009 Apr 2;4:9. doi: 10.1186/1749-799X-4-9. https://pmc.ncbi.nlm.nih.gov/articles/PMC2682792/

Timing of NSAID use is important perioperatively, as the use of indomethacin for the first 7 days after surgery has been shown to contribute to the deterioration of healing compared to treatment after 7 days. Indomethacin has also been shown to have negative effects on the proliferation of human tenocyte cultures.³²

If you have a wound management plan, then all the meds you take should be reviewed for potential negative effects on healing. A lot of the anti-inflammatory herbs we may take in our daily stack also act as coxibs.

However, they may have hundreds of different compounds and modes of action which counteract potential negative effects in a way that allopathic drugs don’t. You need to research the literature and herbal reference books to highlight any red flags.

For instance, berberine is a multi-role therapeutic and is an accessible alternative to ivermectin. It’s also a potent COX-2 inhibitor, with strong anti-inflammatory action.

But does this mean that it therefore impairs healing? Not at all:

Regarding berberine (BBR) and wound healing, this study conflicts with the findings from studies into allopathic coxibs, too:

Omega-3 fish oils are another paradox. They also inhibit COX-1 and COX-2, but through altering the composition of membrane lipids instead of by irreversible binding to amino acid residues and post-translational modification (PTM).11

Even though they inhibit prostaglandins, wound healing is not impaired because they stimulate requisite mechanisms through alternative pathways:

Prior to the prescribing of medication and formulation of a wound management plan, it is essential that a full and complete medication history is taken.

Knowledge about medications with potential to cause wounds or delay wound healing aids in reducing these adverse effects from occurring. Medications with long half-lives may still exert their effects when patients are no longer taking them.

Identifying previously administered medications can help to mitigate potential negative consequences. For example, perioperative wound complication risk can be reduced by delaying surgery after administration of mTOR inhibitors or VEGF inhibitors or withholding NSAIDs perioperatively.

Regular reviews of medications ensures medications with potential negative wound healing impacts are not continued where no longer indicated. Medication dosage and treatment duration also affects the risk of impaired wound healing. Treating an acute gout flare with colchicine for a short duration will have a smaller impact compared to chronic daily dosing for gout prevention.

I’m not saying that this is a binary choice. If you are in severe pain or need to take certain drugs to treat a life-threatening condition, then it may be a trade-off.

One of the reasons for this Substack (and many others) is to help you to give informed consent:

A pharmacist is the ideal professional to consult prior to prescribing a new medication, especially for patients with wounds. Hospital pharmacies have a dedicated drug information service which can be utilised to support patient-centred prescribing.

Determining the risks and benefits associated with medication use is critical. In some cases, medication use is essential and life-saving and potential associated negative impacts may be accepted, such as in cancer or anticoagulation therapy.

Where there are no alternative options available, the choice to use a medication that may impede wound healing may be accepted, but wound prevention and management must be prioritised.

Conflict of interest

The authors declare no conflicts of interest.

More: https://journals.cambridgemedia.com.au/wpr/volume-32-number-1/negative-impact-medications-wound-healing

“Effect of non-steroidal anti-inflammatory drugs on post-surgical complications against the backdrop of the opioid crisis”, from 2018, by Zhao-Fleming et al.,12 further discusses the mechanisms of healing and how NSAIDs disrupt this:

NSAIDs provide anti-inflammatory, antipyretic, analgesic and thrombotic effects through the inhibition of the enzymes cyclooxygenases 1 and 2 (COX-1 and COX-2). These enzymes play a key role in the synthesis of prostaglandin H₂ (PGH₂) from arachidonic acid (AA) [9]. PGH₂ is a precursor for prostaglandins and thromboxanes, collectively referred to as prostanoids, which are synthesized from PGH₂ through tissue-specific isomerases [9].

Some biologically active prostanoids include prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂₎, prostacyclin I₂ (PGI₂), and thromboxane A₂ (TXA₂) [9].

These molecules have diverse functions across a multitude of cell types in vivo. PGE₂ is a pro-inflammatory prostanoid that acts on the kidney, gastrointestinal (GI) mucosa, and the brain.

One of the reasons why NSAIDs can lead to stomach ulcers:

Within the GI tract, PGE₂ promotes mucus secretion, production of bicarbonate, and mucosal vasodilation [10]. When PGE₂ synthesis is inhibited, the GI mucosa is no longer protected from stomach acid, resulting in a higher potential for peptic ulceration and epithelial injury [10].

Your kidney function may be seriously impaired by NSAIDs:

In the renal system, PGE₂ acts as a pre-glomerular vasodilator, allowing for sufficient renal perfusion [9]. If PGE₂ synthesis is limited, the glomerular filtration rate is reduced, leading to reduced renal perfusion, thus creating the possibility for acute kidney injury.

Prostaglandins play a significant role in allergenic responses, as well as in systemic cardiovascular vasodilation (i.e., they help to lower your BP):13

PGD₂ is a prostanoid that is predominantly produced by mast cells and acts on D prostenoid receptors (DP₁ and DP₂) [9]. During an allergic response, PGD₂ mediates a pro-inflammatory response, especially in the lungs, causing vasodilation and bronchoconstriction [9]. PGD₂ has also been shown to inhibit hair follicle neogenesis [11].

PGI₂ works in opposition to TXA₂ to maintain vascular homeostasis:

TXA₂ and PGI₂ are two prostanoids that play significant roles regulating vasodilation and thrombosis within the cardiovascular system [9].

TXA₂ production is primarily controlled by COX-1, while PGI₂ production is mediated by COX-2 [9]. TXA₂ acts a vasoconstrictor and platelet aggregator, while PGI₂ has opposite effects, acting as a vasodilator and inhibitor of platelet aggregation [9]. Together, these two prostanoids maintain vascular homeostasis.

The approved narrative is that NSAIDs can lower your risk of thrombosis, but this isn’t necessarily so:

However, when either COX-1 or COX-2 is inhibited through the use of NSAIDs, vascular complications can arise. When COX-2 is selectively inhibited, PGI₂ production is reduced and the pro-thrombotic activity of TXA₂ is left unbalanced [9]. This can lead to thrombotic injuries such as myocardial infarction, stroke, and deep-vein thrombosis. Because of the many pathways in which NSAIDs are involved, caution must be applied when prescribing them to prevent unwanted side effects.

Wound healing is a complicated, multi-step process, and NSAIDs can disrupt several of these stages:

NSAID animal studies

Effects on wound healing

Proper wound healing requires a strict series of events [12, 13]: hemostasis (vascular restriction and blood clotting), inflammation (clears out dead cells and pathogens), proliferation (including re-epithelization, angiogenesis, collagen synthesis, extracellular matrix formation, and wound contraction) [14], and remodeling (collagen remodeling with vascular maturation and regression).

NSAIDs can disrupt several processes in the proliferation step through inhibition of the COX pathways [15]. COX-coupled prostaglandins, namely PGE₂ and PGD₂, are important in wound healing [16].

NSAID-treated wounds were shown to display decreased keratinocyte proliferation and decreased vascular endothelial growth factor (VEGF) expression. This was confirmed in later studies that demonstrated NSAID-mediated decreases in PGE₂ reduced VEGF expression in keratinocytes [17].

Reduction in PGE₂ levels by the COX2-specific inhibitor, celecoxib, has also been shown to impair wound healing [18]. This deficit is facilitated by a decrease in re-epithelialization through impaired keratinocyte proliferation, reduced angiogenesis, and reduced granulation tissue in the wound.

Decreased myofibroblast differentiation in the wound, along with less wound contraction, was seen in celecoxib-treated mice. NSAIDs also reduced 12-hydroxyheptadecatrienoic acid (12-HHT) production, a ligand for leukotriene B₂ receptor 2 (BLT2), inhibiting healing in mouse corneas [19]. As the 12-HHT/BLT2 pathway is present in the skin [19], this suggests the possibility that NSAIDs may inhibit wound healing through inhibition of this pathway post-skin trauma.

NSAIDs are also involved in reducing levels of inducible nitric oxide synthase (iNOS) [20]. However, the effect of reduced iNOS on wound healing is controversial. Studies have shown a decrease in iNOS inhibits wound healing because of impaired re-epithelialization, specifically impaired keratinocyte proliferation [21].

On the other hand, a celecoxib-induced decrease in iNOS has been shown to increase re-epithelialization through increased myofibroblast proliferation, increased collagen deposition, and decreased skin necrosis [22].

However, the same group showed that this reduction in iNOS caused decreased angiogenesis and decreased tenascin C, which is important in regulating cell proliferation during embryonic development and wound healing [23].

Interestingly, sex differences in response to NSAIDs have also been reported [24], although the data are somewhat inconsistent. While one study showed that female mice, but not males, displayed delayed healing through decreased collagen formation as a result of aspirin use [24], another demonstrated that female mice did not exhibit decreased healing with a selective COX-2 inhibitor [25].

Paradoxically, this research group also reported an increase in VEGF expression in aspirin-treated female mice. While the majority of studies reported thus far have only utilized female mice, a study using male rats found that, although there were fewer fibroblasts in wounds following NSAID use, wound healing was not delayed [26]. Despite these contrasting studies, animal study evidence supporting a causal relationship between NSAIDs and delayed wound healing exists.

NSAIDs may help a type of strep bacteria to infect a wound:

Effects on wound infection

NSAIDs have been implicated in increasing the attachment of opportunistic bacteria, such as group A Streptococcus pyogenes (GAS), to muscle tissue. The clinical association between NSAIDs and GAS has been previously studied and reviewed in detail [27], yet the molecular mechanisms of this association are not yet completely understood. There is evidence that the protein vimentin is transiently expressed on injured muscle post non-traumatic injury, such as in a strain with eccentric contraction [28].

NSAIDs facilitated GAS attachment to the injured muscle and have been hypothesized to slow muscle regeneration, allowing vimentin to be expressed for longer, increasing GAS attachment.

The same group later showed that non-selective NSAID administration increased mortality in mice post-GAS intramuscular challenge [29] and that these non-selective NSAIDs reduced antibiotic efficacy, namely by penicillin and clindamycin.

However, they also saw that neither COX-1 nor COX-2 selective NSAIDs made a difference in mouse mortality or antibiotic efficacy, suggesting that nonselective COX inhibitors may have yet unknown effects through other pathways, and that selective COX inhibitors should be favored in patient groups at high risk for infections.

Other groups have also shown this association. Weng et al. [30] reported that NSAID administration increased wound area of a GAS intramuscular challenge and increased mortality (100% survival in control groups versus 27.5% survival in NSAID group). Upon histological and molecular analysis, they showed that NSAIDs augmented neutrophilic infiltration into the wound and increased interleukin (IL)-6 and tumor necrosis factor (TNF)-α (both pro-inflammatory cytokines) levels, resulting in greater collateral damage to the host.

Overall, the studies on the direct effects of NSAIDs on wound healing are inconclusive with a skew towards inhibiting wound healing. Results are summarized in Table 2.

While there is convincing evidence that NSAIDs inhibit wound healing, there is also evidence that they either have no effect or may even have a beneficial effect by lowering inflammation. However, the effects of NSAIDs cannot be considered in isolation, as perfectly controlled laboratory conditions rarely reflect clinical conditions.

The effects of NSAIDs need to be considered in the context of infectious diseases, where NSAIDs may be promoting certain infections by facilitating bacterial attachment to host cells.

There is less debate about NSAIDs and healing in the world of orthopaedics:

Effects on orthopedic injuries

NSAIDs have long been shunned in the world of orthopedic surgery for their presumed role in the impedance of bony and ligamentous healing. Orthopedic literature based on rat models have proposed that the use of NSAIDs will decrease bone healing and increase rates of the non-union of bones [44].

They then cite many of the studies referred to earlier, which I won’t repeat here.

Overall, these studies show that NSAID use may correlate with the rate of non-union in long-bone fractures. Thus, NSAIDs should be prescribed judiciously in the post-operative period for pain control, and clinicians should bear in mind the possible effects of non-union or mal-union of long bone fractures when prescribing NSAIDs for pain control.

Taken together, larger scale studies are needed to understand the exact effects of NSAIDs on healing, and specifically alternate effects on male and female patients are especially understudied.

More: https://link.springer.com/article/10.1186/s41038-018-0128-x

I won’t reproduce the whole article here, but “Ten top tips: medications that slow wound healing” from the journal “Wounds International” by Black & Hotaling (2023)14 may be another useful reference for you.

Key takeaways:

• 1. Anticoagulants are commonly used and alter early healing.

• 2. Nicotine is a powerful vasoconstrictor.

• 3. Expect significant delays in healing if anti-inflammatory medications are in use. The first six medication classes outlined below all impair the inflammatory phase of wound healing.

• The inflammatory response is often considered the major aspect of healing, and it has been said that when there is no inflammation, there is no healing.

• The inflammatory process is an immediate reaction to the foreign protein on bacteria, damaged cells from trauma and many other causes. The body uses inflammation in response to many conditions.

• In wounds, inflammation protects from further damage from the antigen. Histamine, bradykinin, and prostaglandins are released which open the capillary bed, produce oedema, and isolate the antigen so it can be phagocytised by leukocytes.

• Sometimes inflammation must be controlled to reduce damaging oedema; however, the wound healing will slow. The inflammatory cascade also prompts the remaining three phases of healing, so the impact is long lasting.

• 4. Corticosteroids are the most common anti-inflammatory seen in clinical practice.

• 5. Non-steroidal anti-inflammatory drugs (NSAIDs) significantly impair bone healing. …there is evidence to suggest that long-term use of NSAIDs for managing fracture pain and inflammation carries the risk of impaired bone healing. Many providers add these medications to promote multimodal pain management and reduce the dependence on opiates.

• The introduction of cyclooxygenase (COX)-2 inhibitors, or coxibs, held the promise of providing the same therapeutic benefits as nonspecific NSAIDs with fewer gastrointestinal adverse effects. However, this class of medications has been shown to have potentially serious consequences for the cardiovascular system.

• 6. Anti-rejection/immunosuppressant medications negatively influence the wound‐healing process.

• 7. Immunomodulators increase the risk of serious infections.

• 8. Tumour necrosis factor (TNF) controls many aspects of wound healing.

• 9. Colchicine reduces inflammation and collagen deposition.

• 10. Chemotherapeutic medications inhibit cell proliferation.

• More: https://woundsinternational.com/journal-articles/ten-top-tips-medications-that-slow-wound-healing/

The role of prostaglandin E2 (PGE2) receptor signalling in wound healing

A review from 2021 by Gilman & Limesand reviewed 32 in vitro and in vivo studies which investigated the role of prostaglandins in healing.

It would be more surprising if NSAIDs and other coxibs didn’t have any negative effects, given the complexity of the healing processes and the pivotal role of PGEs.

Key takeaways from “The complex role of prostaglandin E2-EP receptor signaling in wound healing”:15

• PGE₂ signals via four G protein-coupled receptors, termed EP-receptors 1-4 that induce distinct signaling pathways upon activation and lead to an array of different outputs.

• Recent studies examining the role of PGE₂ and EP receptor signaling in wound healing following various forms of tissue damage are discussed in this review.

• Wound healing is a highly complex and coordinated process that has three sequential but overlapping phases:

• 1) hemostasis/inflammation, consisting of vascular changes, immune infiltration, and inflammatory signaling;

• 2) proliferation, focusing on re-epithelialization and angiogenesis; and

• 3) remodeling, aiding in maturation and strengthening of the newly generated tissue (1).

• Following a wounding event, these processes must be tightly regulated to ensure that proper healing occurs.

• Dysregulation of any stage of the response can lead to either insufficient repair or chronic inflammation and excessive tissue damage. Therefore, coordinated inflammation is critical for a wound to heal properly, which requires a number of different cell types and inflammatory signals.

• Inhibiting PGE₂ production with COX inhibitors can be detrimental to wound healing. After mouse ear hole punch injury, treatment with the selective COX-2 inhibitor, celecoxib, reduced wound closure due to decreased macrophage recruitment and M2 macrophage numbers, and reduced blood and lymph vessel formation at the injured site due to decreased transcriptional content of LYVE-1, VEGFR3, VEGF-C, and VEGF-D (21).

• In a rodent model of bone injury, treatment with the nonsteroidal anti-inflammatory drug, naproxen, led to a reduction in serum PGE₂ levels, which correlated with poorer woven bone formation following mechanical loading, indicating a need for PGE₂ signaling in bone remodeling postinjury (30).

• Together, these studies underline the importance of PGE₂-mediated signaling following injury, but more studies defining the role of EP receptors in these responses would be highly beneficial.

• CONCLUSIONS

  Taken together, these studies illustrate that PGE₂ is a crucial eicosanoid in all three phases of the wound healing response.

• Regulated PGE₂ signaling is important for proper repair to occur and treatment with nonsteroidal anti-inflammatory drugs may be detrimental to a healing tissue.

• More clearly deciphering the roles of upstream mediators of PGE₂ production, including PLA₂, COX-1, COX-2, mPGES-1, mPGES-2, and cPGES with future studies will help delinate the inflammatory pathways leading to elevated PGE₂ levels and provide more direct therapeutic targets for modulating PGE₂ production.

Once again, the Big Pharma mantra is to medicate first and to worry about modes of action and possible pathologies at a later date—preferably never:

• Finally, a clearer understanding how EP receptor activation is PGE₂ concentration-dependent across isoforms and tissue types could help guide the use of NSAIDs or 15-PGDH inhibitors to modulate PGE₂-mediated outputs.

• Further evaluation of EP-specific signaling and generation of EP-selective agonists and antagonists may provide novel therapeutic treatment options to enhance wound recovery following an injury.

• More: https://journals.physiology.org/doi/full/10.1152/ajpregu.00185.2020

Do NSAIDs mimic accelerated ageing?

A study found that a protein called 15-PGDH increases with age. The problem with this is that 15-PGDH also inhibits prostaglandin E2, which leads to accelerated degradation of ageing cartilage and joints.

The researchers found that when they injected old animals with a drug that inhibits 15-PGDH, the cartilage in their joints significantly regenerated and was thicker and more functional.

Similar results were found with osteoarthritis prevention after knee injury surgery for ACL tears. This is a common complication that affects about 50% of those with an injured joint within 15 years.16

Inhibition of 15-hydroxy prostaglandin dehydrogenase promotes cartilage regeneration (2025)

Abstract

Aging or injury to the joints can lead to cartilage degeneration and osteoarthritis (OA), for which there are limited effective treatments. We found that expression of 15-hydroxy prostaglandin dehydrogenase (15-PGDH) is increased in the articular cartilage of aged or injured mice.

Both systemic and local inhibition of 15-PGDH with a small molecule inhibitor (PGDHi) led to regeneration of articular cartilage and reduction in OA-associated pain.

Using single cell RNA-sequencing and multiplexed immunofluorescence imaging of cartilage, we identified the major chondrocyte subpopulations.

Inhibition of 15-PGDH decreased hypertrophic-like chondrocytes expressing 15-PGDH and increased extracellular matrix-synthesizing articular chondrocytes.

Cartilage regeneration appears to occur through gene expression changes in pre-existing chondrocytes, rather than stem or progenitor cell proliferation. 15-PGDH inhibition could be a potential disease-modifying and regenerative approach for osteoarthritis.

More (paywalled): https://pubmed.ncbi.nlm.nih.gov/41308124/

3.0 Parting shots

3.1 Supply chain impacts on statins and aspirin

In spite of it being an NSAID, aspirin is, on balance, far more beneficial to health than statins.

Medication shortage hits UK as statins and aspirin ‘disappear from shelves’

Millions of people around the world take medication, and it’s super important they use it correctly at all times. However, recently, the UK and Ireland have been hit with a medicine shortage, and it’s even impacting common medications like statins and aspirin.

A pharmacist, known as Pharmacist Anum, recently drew attention to the issue in a candid video explaining exactly what’s happening, and it’s expert advice worth knowing. With so many people relying on medication to aid their health, it’s a subject they need to be clued up on so they understand what’s going on and why they could experiencing issues at pharmacies.

It's not the first piece of advice she's shared recently either. Just weeks ago, she also drew attention to vital information people need to know about taking statins.

In the clip, Pharmacist Anum said: “Have you recently been told that your medication is out of stock at your pharmacy? We’re having nationwide stock supply issues here at the moment, and let me explain why.

“Unfortunately, what used to be short-term, sporadic stock supply issues are now becoming long-term and regular issues. We’re even seeing common, everyday medications going out of stock, such as aspirin.”

Explaining why this is happening, she added: “Firstly, is global manufacturing issues. So here we rely quite heavily on overseas drug manufacturers to get our medications from.

“If there is an issue with the manufacturers, in say China or India, that can reflect the supply here. Secondly, the UK regulates medication prices, and the NHS often gets medications at a rock bottom price.

“When there’s a shortage of medications, manufacturers are likely to prioritise other countries where they can get more profit from selling their medication. Another issue is Brexit and logistics.

“Ever since Brexit, there have been added regulatory and supply chain pressures. Finally, just an increase in demand for some medications.

“Some medications, such as ADHD medications or weight loss medications, we’re seeing prescribed more than ever. Drug companies may not be able to keep up with the demand, therefore causing delays.”

Sometimes shortages are not just pharmacy-related issues, as they can form a part of a much bigger problem. In fact, it can be global, which is perhaps why some medications appear to be “disappearing from shelves”.

More: https://www.msn.com/en-gb/health/other/medication-shortage-hits-uk-as-statins-and-aspirin-disappear-from-shelves/ar-AA1X71JA

Which leads me to recall an infamous review from 2008 that found that mortality either stays the same or even decreases when doctors go on strike:

Doctors’ strikes and mortality: a review

Abstract

A paradoxical pattern has been suggested in the literature on doctors’ strikes: when health workers go on strike, mortality stays level or decreases.

We performed a review of the literature during the past forty years to assess this paradox. We used PubMed, EconLit and Jstor to locate all peer-reviewed English-language articles presenting data analysis on mortality associated with doctors’ strikes.

We identified 156 articles, seven of which met our search criteria. The articles analyzed five strikes around the world, all between 1976 and 2003. The strikes lasted between nine days and seventeen weeks.

All reported that mortality either stayed the same or decreased during, and in some cases, after the strike. None found that mortality increased during the weeks of the strikes compared to other time periods.

The paradoxical finding that physician strikes are associated with reduced mortality may be explained by several factors. Most importantly, elective surgeries are curtailed during strikes.

In other words, experienced consultants get called to the shop floor:

Further, hospitals often re-assign scarce staff and emergency care was available during all of the strikes. Finally, none of the strikes may have lasted long enough to assess the effects of long-term reduced access to a physician. Nonetheless, the literature suggests that reductions in mortality may result from these strikes.

More: https://pubmed.ncbi.nlm.nih.gov/18849101/

3.2 Caught red-handed: Wes deleting Epstein-related posts

The grift that keeps on giving.

Somewhat embarrassing for the UK’s Secretary of State for Health and Social Care.

Wes has also been named as a possible contender for Prime Minister, and I’m not kidding.

To paraphrase the late, great Sir John Betjeman:

“Come friendly bombs and land on Westminster! It isn't fit for humans now.”

Desperate Streeting tries to distance himself from Mandelson

Sentient forehead and Labour big man Wes Streeting has come out swinging for Mandelson. Specifically, he’s told the BBC that the key issue is people failed to believe Epstein’s victims.

The problem is that the people who didn’t believe the victims includes the government Streeting is a part of. After all, why would they have promoted Mandelson otherwise, when they knew about his connections to Epstein?

Streeting — the allegations

Don’t get me wrong, it’s wonderful that Streeting has managed to pull himself away from privatising our NHS to stand up for the victims, but all is not as it seems.

Streeting’s Twitter/X history shows that he was silent on Epstein until this morning. This was despite years of actual women coming forward, and the known links between Mandelson and Epstein.

Additionally, it seems Streeting has only mentioned Mandelson himself a few times at first glance. But that doesn’t factor in the tweets he’s recently deleted:

It appears Streeting is trying to keep his relationship with Mandelson quiet. When Streeting was running for office, it appears Mandelson joined him on his campaign trail. Not only that, but he joined the “Sunday Supper Club”, which was an exclusive group Mandelson frequently attended.

‘Protect women’ is political posturing

With no previous mentions of Epstein, this seems to be political posturing. And at a time when Starmer’s position is weakened as prime minister, this is obviously a transparent attempt to undermine him in preparation for a future leadership challenge.

More: https://www.thecanary.co/opinion/2026/02/04/streeting-tries-to-distance/

4.0 Concluding remarks

Don’t take any medication (including herbals, vitamins and minerals) for an indefinite period without seeking professional advice, either in person from a doctor or pharmacist, or from studying clinical and other research reports.

If you have an injury that just doesn’t seem to get better, it may not be due to age, or stress, or because you have “poor healing flesh”; it may be due to the meds you are taking.

If you self-medicate, consider taking a break from time to time or rotating your stack, but don’t stop taking any prescribed medications without taking professional medical advice first.

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5.0 Disclaimer

This site is strictly an information website reviewing research into potential therapeutic agents. It does not advertise anything or provide medical advice, diagnosis, or treatment. This site does not promote any of these as potential treatments or offer any claims for efficacy. Its content is aimed at researchers, registered medical practitioners, nurses, or pharmacists. This content is not a substitute for professional medical advice, diagnosis, or treatment.

Always seek the advice of your physician or other qualified health provider with any questions you may have regarding a medical condition. Never disregard professional medical advice or delay in seeking it because of something you have read on this website.

Always consult a qualified health provider before introducing or stopping any medications, as any possible drug interactions or effects will need to be considered.

Any extracts quoted in the previous article are for non-commercial research and educational purposes only and may be subject to copyright from their respective owners.

6.0 References

Reading time:

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Any extracts used in the following article are for non-commercial research and educational purposes only and may be subject to copyright from their respective owners.

Contents

1.0 Introduction

2.0 Discussion

• 2.1 History of analgesics

• 2.2 An overview of acetaminophen toxicity

  • Acute Hepatic necrosis

  • Dosing and pathophysiology

• 2.3 Glutathione Depletion

  • Time course of depletion

  • Glutathione depletion and DNA damage

  • Dietary sources of phytonutrients for glutathione support

• 2.4 Acetaminophen and immune response

  • Liver injury

  • Acetaminophen and cancer

• 2.5 Acetaminophen and harm to the child

3.0 Parting shots

• 3.1 Flu vax failure… again

• 3.2 Wes is back, and boy, is he in trouble

4.0 Concluding remarks

5.0 Disclaimer

6.0 References

1.0 Introduction

Not needing much of an introduction, Acetaminophen (C8H9NO2) is the generic name of an ingredient of many well-known brands, including Paracetamol, Tylenol, Panadol, Aceta, and Tylenol Extra Strength.

The original patent for "paracetamol” expired in 2007,1 but newer patents exist with later expiration dates for specific formulations, dosages, and combination products.

It is used systemically as a treatment for a wide range of conditions, including:2

• Back Pain

• Chiari Malformation

• Chronic Pain

• Dengue Fever

• Eustachian Tube Dysfunction

• Fever

• Headache

• Muscle Pain

• Neck Pain

• Pain

• Plantar Fasciitis

• Sciatica

• Transverse Myelitis

It’s also useful for reducing the amount of opioids needed after surgery, for example, post-caesarean (Ng et al., 2019)3 and oncologic breast surgery (Alsaadi et al., 2024).4

The global market for acetaminophen is huge. In 2024, it was valued at $10.6 billion, and is expected to appreciate to $14.1 billion by 2030, at a compound annual growth rate (CAGR) of 4.8%.

Upstream active pharmaceutical ingredient capacity is concentrated in a handful of Asia-based chemical manufacturers, whereas downstream finished branded dose production is spread across a range of multinationals and private-label companies.

The pediatric market is exploited by providing acetaminophen in liquid formats, with dosing syringes and child-resistant caps.

Oral administration accounts for 87% of global revenues. Rectal and intravenous use accounts for most of the balance.5

The purpose of this Substack is not to advise you whether to take the product or not; it is so that, whatever your choice, you have informed consent. As ever, I consult representative peer-reviewed literature or preprints and present the research findings.

Thus, this is not a work of opinion; it aims to stick objectively to facts gained through the scientific method.

That doesn’t mean that these are definitive; such findings are always open to discussion, and further research is often warranted. But if such questions are unresolved, the precautionary principle should apply.

Emphasis is mine in bold, and some passages are lightly reformatted for legibility on portable devices.

2.0 Discussion

2.1 History of analgesics

The forerunners of acetaminophen were discovered by accident. Phenacetin provided a convenient way for drug manufacturers to consume cheap by-products from dye manufacturing. It proved to be a popular, ill-fated way to meet Swiss watch production targets. Acetaminophen was thought, mistakenly, to be a much safer alternative. That perception remains to this day.

Brune et al. (2014) provide an excellent reference piece:

Acetaminophen/paracetamol: A history of errors, failures and false decisions

1. Discovery by error and serendipity

1.1 Acetanilide

In 1884, an important discovery took place in Strasbourg (Germany, later France). At that time, the medical clinic of Strasbourg was headed by the famous professor Adolf Kussmaul. Two young assistants, A. Cahn and P. Hepp, asked his advice on how to treat a patient who suffered from many ailments, among those fever and worm infestation (Cahn and Hepp, 1886).

Kussmaul suggested to try naphthalene, which had been recommended as an intestinal antiseptic (Sneader, 1985). The two young physicians followed the advice and were disappointed by the result – most ailments remained, including the worms. Much to their surprise, they observed, however, that the fever of the patient fell shortly after the administration of the putative naphthalene.

The two physicians wondered why the antipyretic effect had not been described before. Researching the source of the ‘naphthalene’, they learnt that (the local) pharmacy had provided them with acetanilide (Fig. 1) instead of naphthalene – an error with long-lasting consequences!

Consequently, Cahn and Hepp tested acetanilide in rabbits and dogs (at that time, an animal ethics request was not needed) (Cahn and Hepp, 1886). They observed reliable but short-lasting antipyretic activity. Both species did not suffer from (visible, acute) toxicity. The young physicians then dared to try acetanilide in 24 patients (certainly without informed consent). They were excited as all of them showed a reduction of fever. Acetanilide turned out as effective as phenazone, which had been successfully put to the marketplace two years before (Filehne, 1884).

A metaphor for Big Pharma dumping toxic by-products on the market:

1.2 Phenacetin

It is to be credited to O. Hinsberg (1913) to have synthesized phenacetin (Fig. 1). Together with a pharmacologist from Freiburg, A. Kast, he showed that the new compound worked as acetanilide but was less toxic (Hinsberg, 1913). In addition, it could be produced at low costs, as Bayer and other companies were flooded with large amounts of para-nitrophenol, which is a by-product of the production of dianisidine (needed to produce the new synthetic ‘aniline dyes’).

C. Duisberg (1913, director of Bayer) reported that Bayer was searching for customers of the useless para-nitrophenol. He states that it was pure luck (Duisberg, 1913) that O. Hinsberg together with A. Kast found that phenacetin can be produced easily from para-nitrophenol and that it is both more effective and safer than acetanilide.

Paracetamol was the main active metabolite of phenacetin and was responsible for its analgesic effects:

The situation changed in 1946 when American scientists reinvestigated the phenacetin metabolism. Lester and Greenberg at Yale together with Flinn, Brodie and Axelrod (Axelrod, 1988) (Fig. 3), in New York, showed that paracetamol was the main (and active) metabolite of phenacetin, a claim originally put forward by Hinsberg and Treupel (1894).

Brodie believed that paracetamol is the safer and more active drug as compared to phenacetin, as phenacetin could lead to aniline, but paracetamol not (Flinn and Brodie, 1948). Still, paracetamol was not the end of the metabolic cascade. It was eliminated as glucuronide or sulfate, but some paracetamol was oxidized to NAPQI (N-acetyl-p-benzoquinone imine), a highly reactive compound, which we will have to deal with later.

Methemoglobin toxicity: a condition where excessive methemoglobin in the blood prevents red blood cells from delivering oxygen to the body, leading to symptoms like cyanosis (blue skin), fatigue, headache, and, in severe cases, seizures, coma, or death.

Anyway, from the data accrued by the Axelrod group, it was plausible that paracetamol (in the United States, acetaminophen) could be the better drug, free of methemoglobin production, but retaining the full analgesic/antipyretic power of phenacetin. Regrettably, this conclusion was partially false.

These observations supported the market introduction of paracetamol/acetaminophen. Interestingly, the comparatively small problem of methemoglobin production was remedied, but the liver toxicity of paracetamol was overlooked until 1966 (Davidson and Eastham, 1966).

The metabolization of phenacetin to paracetamol is slow, allowing for the detoxification of most of the emerging paracetamol (Fig. 1) into harmless glucuronides or sulfates. Only small amounts of the toxic, reactive NAPQI are produced from phenacetin (Fig. 1).

The introduction of paracetamol led to a re-evaluation of the toxicity of phenacetin. This was a very timely as a frightening observation in Switzerland hinted at serious kidney problems from the intensive use of phenacetin.

1.4 Phenacetin, the culprit of the first side-effect epidemy?

Relieve of pain and fever was achieved by the use of the new synthetic, antipyretic analgesics. Aspirin (introduced 1899), phenazone (1884) and acetanilide, later substituted by phenacetin (1887), dominated the drug market during the first 50 years of the 20th century. They were the most widely used and prescribed drugs in most countries (and they still are). All of them were found by serendipity, often under false assumptions.

The attempts for further improvement led to the use of pharmacological analogues. In the United States, United Kingdom and Australia, aspirin and other derivates of salicylic acid were the standard analgesics. In continental Europe and parts of Asia, phenazone and derivates (aminophenazone, dipyron) were the prevailing drugs. When the major metabolite of phenacetin, paracetamol, was introduced (1953), few consumers switched to the new compound, but the situation changed soon.

Firstly, the economic situation of Europe and the United States improved and the demand for consumer goods with it. In Switzerland, the traditional watch industry gained momentum. New mechanical watches were assembled under time constraint (job work), mostly by women.

The job work led to headache and exhaustion after a few hours of assembling small and delicate mechanical watches (Moeschlin, 1957, 1958; Dubach et al., 1975, 1991).

Ah, the wonder of modern medicine: spiked bread and butter.

An effective method to overcome pain and fatigue was a break, coffee and a piece of bread covered with butter and punched into the butter ‘headache pills’.

Most prominent was a brand produced by Hofmann-La Roche, called Saridon®. [At that time, Saridon® contained caffeine, phenacetin, propyphenazone and barbiturates (Zollinger, 1955).]

At the same time, it was customary in Switzerland that elderly women had their coffee in one of the nice coffee shops. They supplemented their coffee with a couple of headache pills, which they could get directly from the waiter. These and other habits, not only in Switzerland, increased the use of headache pills, which typically consisted of the three ingredients: phenacetin (or phenazone, propyphenazone), aspirin and caffeine. Nobody was afraid of any harm as all the components and the mixtures of them were regarded safe, useful and pleasant – a major error as turned out later.

It is the merit of scientists from Switzerland (Fig. 4) who observed an increase in incidence of interstitial nephritis in the population of Basel and nearby regions. They described the pathology of this ‘disease’ (better: chronic intoxication).

It occurred often in young (30–50 years old) women (many workers in the watch industry) who suffered initially from mild hypertension, later from interstitial nephritis going along with pigmentation of the skin and early death from cardiac infarction and/or cardiac insufficiency.

Initial investigations excluded phenacetin as the cause. No change there (!)

Spühler and Zollinger (1953) speculated about a change in the environment, including the widespread use of barbiturates and antibiotics (Spühler and Zollinger, 1953). Moreover, they realized that in most cases the disease started with headache, which was at first episodic and later almost permanent. Today, we call it ‘rebound headache’ (Rapoport et al., 1996). Many of the patients were suffering from this type of chronic headache and ingested headache pills (often Saridon®) regularly – without much relieve.

Eventually, a different physician correctly connected the cause with effect, but it took until 1980 for the paradigm shift to occur:

In contradistinction to what is written in many articles, in the original paper, Spühler and Zollinger (1953) did not suspect a causal relationship between the use of analgesics and interstitial nephritis, although one-third of their (first) 44 patients had consumed analgesics regularly.

It was the merit of a clinician, S. Moeschlin (Fig. 4), to pinpoint the relation between ‘Saridon’-abuse and kidney decay (Moeschlin, 1957, and quoted in ‘Der Spiegel’, July 1958). It was only shortly after their publication that a worldwide increase of interstitial nephritis was observed and a connection between the use of analgesics (mostly combinations) and nephropathy (analgesic nephropathy, phenacetin nephritis, Saradon nephritis) was postulated (Nanra, 1980; Kincaid-Smith, 1981; Dubach et al., 1991).

Exit phenacitin, enter paracetamol, the safe new wonder drug:

The observation from Switzerland and many other countries indicated that phenacetin was the major culprit of interstitial nephritis, hypertension and death. Consequently, beginning in the 1950s of the last century, paracetamol supplanted phenacetin. It was hoped that the abuse potential would be lower and the kidney toxicity absent.

There was indeed some reduction of the ominous interstitial nephritis, but the coincidence of the reduction may rather reflect the intensive attempts, on the one hand, to hinder consumers to abuse combination analgesics and, on the other hand, to get those who were habitually consuming these compounds, deconditioned and detoxified (Drukker et al., 1986; Feinstein et al., 2000).

It turned out again that combination analgesics taken regularly not only lost their anti-headache activity but rather induced headache attacks (Rapoport et al., 1996). In the end, it appeared that kidney problems were solved and paracetamol could be used as a safe and active analgesic, even in pregnant women and children. Regrettably, this assumption was wrong again.

Paracetamol does not cause methemoglobin production (Prescott, 1980) but it turned out to be hepatotoxic due to its very reactive intermediate (NAPQI, Fig. 1), which is produced in the liver and kidney cells via the CYP P450 metabolizing enzymes, preferentially in the liver and kidney. Hundreds of death cases and thousands of hospital referrals result from overdosing, voluntarily and accidentally every year (Moynihan, 2002; Lee, 2004).

More: https://onlinelibrary.wiley.com/doi/abs/10.1002/ejp.621

2.2 An overview of acetaminophen toxicity

Its danger lies in a successful long-running marketing campaign that, falsely, leads to its perception as being safe. It’s also an ingredient in a wide range of over-the-counter home remedies for colds, ‘flu, aches and pains.

It is relatively easy to accumulate a toxic dose, particularly if you are feeling low and taking multiple medications. The likelihood of this increases due to “acetaminophen rebound headache”, caused by tolerance due to frequent use of pain medication. “Frequent” may mean taking it for as little as 2-3 days a week.

Medication overuse headaches tend to happen to you every day, or nearly every day, and may be so intense that they wake you from sleep.6

If your liver and kidneys are not young and healthy, your threshold for toxicity will be lower still, with alcohol or other painkillers further compounding the effects.

Acute Hepatic necrosis

Fortunately, the liver is unique in that it is the only internal organ with the remarkable ability to regenerate itself with new, functional cells after sustaining cell loss through injury or necrosis. But you must eliminate the toxic agent before the damage is too extensive.

Description. The clinical course of acute hepatic necrosis resembles an acute, toxic injury to the liver with sudden and precipitous onset, marked elevations in serum aminotransferase levels, and early signs of hepatic (or other organ) dysfunction or failure despite minimal or no jaundice. Rapid recovery after withdrawal of the agent is also typical.

Other organ failure, such as lung, kidney or bone marrow, may also be present and may overshadow the hepatic injury. Acute hepatic necrosis is typically caused by a direct hepatotoxin and is usually dose dependent and “expected”, rather than idiosyncratic.

Latency to Onset. The time to onset of acute hepatic necrosis is typically short, 1 to 14 days.

Symptoms. Usually abrupt onset of nausea, weakness, fatigue and abdominal pain; somnolence and mental clouding may occur early. Itching is rare and jaundice appears later. Other organ failure (kidney, lung, bone marrow) may be prominent.

Serum Enzyme Elevations. ALT rising rapidly to above 800 IU/L (>20 times ULN), with alkaline phosphatase <2 times ULN (<230 U/L). As a result, the R ratio (ALT divided by Alk P, both expressed as multiples of the upper limit of normal) tends to be very high (>10).

If measured, LDH levels are also elevated. Serum creatinine, BUN, INR and ammonia are also often raised, sometimes before hyperbilirubinemia (which is usually mild to moderate even at the height). Strikingly, the serum enzymes usually improve rapidly with stopping the medication, falling by 50% within 8 days.

More: Acute Hepatic Necrosis (2019)

A StatPearls post by Agrawal et al. (last update: 2025) provides some alarming stats.

Key takeaways from “Acetaminophen Toxicity“:7

• Although relatively safe at therapeutic doses, acetaminophen poisoning causes hepatic necrosis.

• Acetaminophen toxicity is the second most common cause of liver transplantation worldwide and the most common cause of liver failure in the United States.

• Responsible for 56,000 emergency department visits and 2600 hospitalizations, acetaminophen poisoning causes 500 deaths annually in the United States.

• Notably, around 50% of these poisonings are unintentional, often resulting from patients misinterpreting dosing instructions or unknowingly consuming multiple acetaminophen-containing products.

• The mortality associated with acetaminophen overdose is low if recognized and treated within the first 8 hours.

• Factors such as alcohol use, genetics, age, medications, herbal supplements, and nutritional status can enhance acetaminophen’s ability to damage the liver.

• In addition to liver failure, affected patients may experience renal failure as a consequence of acetaminophen toxicity.

• Acetaminophen toxicity is prevalent, primarily due to the drug’s widespread availability and the common perception of its safety.

• In the United States, acetaminophen toxicity is the leading cause of liver failure. Over 60 million Americans use acetaminophen weekly, often unknowingly, due to its presence in various combination products, particularly those containing opioids and diphenhydramine.

Disturbingly, its specific mode of action is still not fully understood. If you don’t know how it works, how can you predict its long-term toxicology, drug interactions, or our genetic susceptibility?

Cyclooxygenase (COX): An enzyme that creates prostaglandins and thromboxanes from arachidonic acid (an animal-sourced omega-6 fatty acid). Two isoforms, COX-1 and COX-2 are targets of nonsteroidal anti-inflammatory drugs (NSAIDS). COX-1 was thought to produce homeostatic prostaglandins (PGs), and COX-2 PGs were thought to be pathophysiological (linked to pain and inflammation), but it’s not that clear-cut.8

Prostaglandins (PGs): Hormone-like substances produced in most tissues that play a role in bodily defence, natural physiology, inflammation, pain, and fever.

Thromboxanes: A group of lipids involved in blood hemostasis, especially for clot formation through platelet aggregation.

• Although its mode of action, specifically the selective inhibition of cyclooxygenase in the brain, is not fully understood, acetaminophen effectively alleviates fever and pain.

• In addition, it may inhibit prostaglandin synthesis in the central nervous system and directly affect the hypothalamus to produce an antipyretic effect.[4]

• If left untreated, acetaminophen toxicity can lead to both fatal and non-fatal hepatic necrosis. Timely intervention is essential in preventing fulminant liver failure and the need for liver transplantation.

• Activated charcoal should be administered within the first hour and N-acetylcysteine within the first 8 hours.

• The most reliable predictor of toxicity is correlating the time of ingestion with the serum acetaminophen concentration using the Revised Rumack-Matthew nomogram. Patients whose levels fall above the treatment line at 4 hours require N-acetylcysteine.

• A striking 50% of cases result from unintentional overdoses, highlighting a crucial need for healthcare professionals to properly educate patients regarding the proper dosing of acetaminophen and its presence in prescribed and over-the-counter preparations.

Dosing and pathophysiology

In the UK, since September 1998, a general sales outlet (such as a supermarket) can only sell paracetamol in a maximum pack size of 16 x 500mg, and a maximum of 2 packs in any one transaction (16 g in total from one outlet). A pharmacy may sell you packs of 32.

Overdoses lead to 100-200 deaths per year in the UK, and it is thought that the change in legislation reduced overdose deaths by 43% per year.9

According to StatPearls, you can still take a toxic dose in 24 hours, even with these pack size limits:

• Acetaminophen is available in immediate-release and extended-release formulations. The dosage forms available are capsules, elixir, gel, oral and intravenous solutions, liquid, tablets, suppositories, syrup, and dissolve packs. When taken at therapeutic doses, acetaminophen has a good safety profile. The therapeutic doses are:

  • 10 to 15 mg/kg/dose in children every 4 to 6 hours with a maximum dose of 80 mg/kg/d

  • 325 to 1000 mg/dose in adults every 4 to 6 hours, with a maximum daily dose of 4 g/d

• Toxicity is likely to develop in adults at:

  • >12 g over a 24 hours

  • 7.5 to 10 g in a single dose

  • Doses >350mg/kg

• Toxicity in children occurs following a single dose of 150 mg/kg or 200 mg/kg in otherwise healthy children aged 1 to 6.

• Acetaminophen is fully absorbed in the duodenum within 2 hours of ingestion. Foods and medications that decrease gastric emptying can delay absorption.

• The liver metabolizes 89% of acetaminophen, leading to saturation of the sulfation and glucuronidation pathways and depletion of hepatic glutathione stores.

• The result is the production of a toxic, highly reactive compound that causes a cascade of oxidative hepatocyte injury.

If you suspect that you have taken too many, time is of the essence.

N.B. These figures are disturbing, another example of regulatory capture.

• Epidemiology

  Acetaminophen toxicity accounts for 50% of all reported cases of liver failure in the United States and 20% of all liver transplants. If discovered and treated quickly, the associated morbidity and mortality are low. Once liver failure develops, the mortality increases to 28%, with one-third requiring a liver transplant.

• Although acetaminophen poisoning is more common in children, adults are more likely to develop hepatotoxicity. Acetaminophen poisoning is responsible for 56,000 emergency department visits, 2600 hospitalizations, and 500 deaths annually in the United States, with 50% of these cases being unintentional overdoses.[5][6][7]

• Approximately 30,000 pediatric acetaminophen poisoning cases are reported to the National Poison Data System annually.

• Factors that increase the risk of hepatotoxicity include underlying liver disease, alcohol use disorder, and unintentional overdose.

• Combination opioid prescriptions containing acetaminophen are an additional risk contributing to acetaminophen poisoning. The United States Food and Drug Administration now requires a lower dose of acetaminophen in all combination products.

• Pathophysiology

  Acetaminophen is rapidly absorbed from the gastrointestinal tract and reaches therapeutic levels in 30 minutes to 2 hours. Overdose levels peak at 4 hours unless other factors are included, such as the co-ingestion of an agent that slows gastric motility or if the acetaminophen is in an extended-release form.[8][9]

The more damage the liver sustains, the longer it takes to eliminate a toxic dose:

• Acetaminophen has an elimination half-life of 2 hours but can be as long as 17 hours once hepatic injury develops. In the liver, acetaminophen conjugates to nontoxic, water-soluble metabolites that are excreted in the urine.[9]

• Metabolism

  Approximately 90% of acetaminophen is metabolized in the liver by the hepatic microsomes via sulfotransferase and UDP-glucuronosyl transferases, forming sulfate and glucuronide conjugates that are excreted in the urine.[10][11]

• About 8% of acetaminophen is metabolized by hepatic cytochrome P450 subfamilies—CYP2E1, CYP1A2, and CYP3A4—through the mixed-function oxidase pathway, generating a toxic, highly reactive, electrophilic intermediate N-acetyl-p-benzoquinoneimine (NAPQI).

• The remaining 2% of acetaminophen is excreted unchanged in the urine.

• At therapeutic doses, acetaminophen produces minimal amounts of NAPQI, which swiftly conjugates with hepatic glutathione to produce nontoxic cysteine and mercaptate compounds excreted in the urine. However, in cases of acetaminophen toxicity, an increased production of NAPQI occurs, depleting hepatic glutathione stores.

Glutathione: “a tripeptide (cysteine, glycine, and glutamic acid) found in relatively high concentrations in many bodily tissues. It plays a pivotal role in reducing oxidative stress, maintaining redox balance, enhancing metabolic detoxification, and regulating the immune system.”10

NAPQI binds with hepatic mitochondrial proteins. The impairment of the mitochondrial electron transport chain (ETC) leads to the generation of reactive oxygen species (ROS), such as superoxide. This activates cell death signalling pathways (the c-Jun N-terminal kinase, JNK, pathway), ultimately causing hepatic necrosis.

• NAPQI gains an additional aryl group and binds covalently to cysteine groups on hepatic macromolecules, particularly mitochondrial proteins, forming NAPQI-protein adducts. The process initiates an irreversible cascade. The results of NAPQI-protein adduct formation include:

  • Oxidative hepatocyte injury

  • Alteration of the mitochondrial ATP-synthase alpha-subunit

  • Hepatocellular centrilobular necrosis

• Processes that contribute to the propagation of hepatic injury include:

  • Generation of nitrotyrosine adducts within mitochondria by toxic free radicals, such as peroxynitrite

    • Results in damage to mitochondrial DNA and ATP-synthase, halting ATP synthesis

  • Lipid peroxidation and membrane damage

  • Release of cytokines, apoptosis-inducing factor, endonuclease G, and reactive nitrogen and oxygen species from damaged mitochondria

    • Causes a secondary inflammatory response

  • Recruitment of inflammatory cells through the innate immune system by damage-associated molecular pattern (DAMP) products, such as nuclear fragments and mitochondrial DNA

    • Typically manifests during clinical stage II

Subclinical toxicity may develop following repeated therapeutic doses due to the following factors:

• Acute alcohol ingestion.

• Chronic alcohol ingestion.

• Chronic liver disease.

• Concurrent medications and herbal supplements: Medications that induce CYP2E1 enzymes, such as carbamazepine, or compete for glucuronidation pathways, such as trimethoprim-sulfamethoxazole and opioids, increase the risk of hepatotoxicity. Herbal supplements such as St John wort, garlic, and germander may enhance the CYP450 activity.

• Nutritional status: Hepatic glucuronidation depends on liver carbohydrate reserves. Fasting or malnutrition decreases the glucuronidation of acetaminophen, causing an increased production of NAPQI.

• Age: The risk of hepatotoxicity increases with age. Children younger than 5 appear less susceptible to hepatotoxicity unless they receive repeated supratherapeutic doses or acetaminophen. The increased supply and regeneration of glutathione and greater activity of conjugation enzymes in children are likely reasons for the lower risk of hepatotoxicity.

• Genetics: The link to genetic polymorphisms that cause acetaminophen’s diminished or excessive oxidative metabolism is unclear. Patients affected by Gilbert syndrome are at risk for increased toxicity.

  

• Tobacco use: Tobacco use induces CYP1A2 enzymes and is an independent risk factor for mortality after acetaminophen overdose. The highest mortality is associated with patients who smoke and drink alcohol.[13]

• Serum phosphate: Low serum phosphate levels result in decreased acetaminophen toxicity, likely due to enhanced cell regeneration supported by hepatocyte phosphate uptake to regenerate ATP.

If you’ve overdosed, you may be asymptomatic for up to 24 hours. It may take 1-4 days before liver and kidney damage lead to severe symptoms.

10-50% of patients with significant liver damage may also develop acute renal failure.

• The clinical course of acetaminophen toxicity consists of 4 stages.[15]

  Stage I

  • First 30 minutes to 24 hours

  • Possibly asymptomatic

  • May experience nausea, vomiting, diaphoresis, pallor, lethargy, and malaise

  • Laboratory values are generally normal, but aminotransferase levels may increase within 8 to 12 hours

  • Central nervous system depression and elevated anion gap metabolic acidosis are possible with a >30 g ingestion

  Stage II

  • 24 to 72 hours

  • Laboratory results reveal hepatotoxicity and nephrotoxicity

  • Clinically, the patient appears improved, but laboratory values worsen

  • Right upper quadrant pain and hepatomegaly

  • Evidence of abnormal prothrombin time (PT) and total bilirubin, along with oliguria and renal function abnormalities

  • Case reports indicate acute pancreatitis

  Stage III

  • 72 to 96 hours

  • Aminotransferase levels peak, with values exceeding 10,000 IU/L

  • More pronounced symptoms, including jaundice, confusion, hyperammonemia, abnormal aminotransferases, and bleeding diathesis, along with recurrence of symptoms from stage 1

  • Acute renal failure in 50% of patients with frank liver failure and 10% to 25% of patients with significant liver damage

  • Indirect hyperbilirubinemia

  • Prolonged PT

  • Hypoglycemia

  • Lactic acidosis

  • The most common stage for death to occur due to multisystem organ failure

  Stage IV

  • If the patient survives stage 3

  • Recovery phase

  • Typically begins by day 4 and completes by day 7

  • Symptoms and laboratory values may require several weeks to normalize

  • Histopathologic changes in the liver become evident

  Acute renal failure due to acute tubular necrosis manifests as an elevated blood urea nitrogen and creatinine, proteinuria, hematuria, and granular and epithelial cell casts on urinalysis. The incidence is less than 2% of all patients, 5% of cases with liver involvement but no hepatic failure, 10% of severe poisonings, and nearly 53% of cases with acute hepatic failure. The renal function generally returns to normal in 1 to 4 weeks. Some patients may require dialysis during this time.

In terms of treatment, activated charcoal (AC) may be given if the patient presents within 1-4 hours after ingestion. The powerful antioxidant N-acetylcysteine (NAC) is given to patients with any evidence of liver damage and acetaminophen ingestion. It may be beneficial for the first 24 hours after ingestion. After this period, hemodialysis and kidney dialysis may be necessary.

• High-Risk Ingestion

  High-risk ingestion consists of a dose of >30g. These patients may require intubation to protect their airway, intravenous fluids and vasopressors, and bicarbonate for metabolic acidosis. Patients with a high-risk ingestion are at an increased risk of developing fulminant liver failure. Activated charcoal should be administered even if beyond 4 hours, and N-acetylcysteine should be initiated immediately. In addition to N-acetylcysteine, hemodialysis is indicated in patients with an acetaminophen concentration >900 mcg/mL.

• N-acetylcysteine Management

  N-acetylcysteine protects against liver toxicity if administered within 8 hours of acetaminophen ingestion. The effectiveness remains unchanged whether initiated between 0 and 4 hours or 4 and 8 hours. N-acetylcysteine acts by preventing the binding of NAPQI to hepatic macromolecules by acting as a substitute for glutathione, serving as a precursor for sulfate, and reducing NAPQI back to acetaminophen.

The prognosis is generally good, provided you are quickly administered NAC:

• The incidence of hepatotoxicity for patients treated with N-acetylcysteine within 8 hours of acetaminophen ingestion is less than 10%, but it increases to approximately 40% when delayed beyond 16 hours.

• When treated promptly, the mortality associated with acetaminophen toxicity is less than 2%. However, if patients present late and develop severe liver failure, mortality is high.

• Approximately 1% to 3% of patients with severe liver failure require liver transplantation.[19][35][36] In general, children younger than 6 have a better prognosis than adults, primarily due to their greater capacity to detoxify acetaminophen.

From the paper by Brune et al. (2014),11 we also learn that even standard doses lead to liver toxicity:

• NAPQI may, in e.g., very slim undernourished persons or alcoholics, cause acute liver decay and death from standard doses (Moore et al., 2013).

• Even healthy young volunteers ingesting standard doses of paracetamol show an increased release of transaminases – a sign of liver toxicity (Watkins et al., 2006).

• This liver toxicity was not seen with phenacetin. Indeed, several researchers wondered if by eliminating phenacetin from human use, ‘the wrong pig’ was killed.

Brune et al. discussed how paracetamol is linked to renal toxicity, asthma, and unexplained liver damage in both adults and children:

• Metabolites from paracetamol–GSH conjugates have been implicated in renal toxicity. Paracetamol–cysteine conjugates aggravated paracetamol-induced renal injury (Trumper et al., 1996; Stern et al., 2005).

• Recent experimental findings provide evidence for a signalling role of NAPQI produced in murine lungs following administration of low non-toxic doses of paracetamol (Nassini et al., 2010).

• NAPQI activated the transient receptor potential ankyrin-1 (TRPA1) expressed by primary sensory neurons, where it stimulated the release of the pro-inflammatory neuropeptides.

• Paracetamol induced neurogenic inflammation in the murine lung. This effect was absent in paracetamol-treated TRPA1-deficient mice. Therefore, NAPQI may also act as a signalling molecule contributing to the asthma-inducing effect of paracetamol observed in several epidemiological studies (for review, see Henderson and Shaheen, 2013).

• Paracetamol is eliminated quickly [t_50%: ∼1 h, in children 1 (newborn) – 8 h]. This made it difficult to provide proof that a patient suffering from acute liver decay has ingested paracetamol (James et al., 2006). This situation changed when several research groups could show that paracetamol is bound covalently to cysteine molecules in different proteins. These acetaminophen/protein adducts remain in the body for several days. They can be detected with chromatographic techniques (Madsen et al., 2007).

• Consequently, the proof that paracetamol has contributed to an acute liver damage has become easier (James et al., 2006, 2013; Heard et al., 2011; McGill and Jaeschke, 2013).

Even healthy individuals taking a standard dose, with normal levels of glutathione, develop some NAPQI toxicity:

• On the contrary, this technique used in healthy patients demonstrates that even at normal doses paracetamol adducts are produced, which, in turn, requires the production of NAPQI that obviously is not completely detoxified by conjugation with glutathione.

• The adducts of paracetamol allow to speculate about the contribution of paracetamol ingestion to otherwise unexplained liver damage. Indeed, it could be shown that in many unexplained intoxications in children (James et al., 2006) and adults (Heard et al., 2011; Khandelwal et al., 2011), paracetamol may have played a major role, as even days after hospital admission protein adducts were found.

It is a danger to the foetus and newborns, as paracetamol freely crosses the placental barrier:

• Moreover, these protein/paracetamol adducts may be produced in embryos and newborns since the necessary enzymes are active in the fetus and paracetamol crosses the placenta barrier freely (Rollins et al., 1979; Byer et al., 1982; Heard et al., 2011).

I’m not going to get into the politics of this, as paracetamol poisoning of children may have been raised as a limited hangout to deflect attention away from the all-too-real vaccine poisoning and autism epidemic.

All sources of toxicity are equally important—from vaccines to acetaminophen to HPV vaccines to valproate to statins, as well as the other allopathic nightmares I’ve been researching. Excuse the pun: “A plague on all their houses”.

• These observations gain importance when seen in context with the multitude of epidemiological and experimental results indicating long-term problems in children who were exposed to paracetamol (and NAPQI) during pregnancy.

• Paracetamol has lost the glamour of being well tolerated and innocuous to pregnant women and newborn children. Large analyses of databanks and case–control studies show that the use of paracetamol during pregnancy may increase the incidence of asthma in children of mothers having used paracetamol during pregnancy (for review, see McBride, 2011).

• Ibuprofen, investigated head to head with paracetamol in a prospective study, was not associated with an increased incidence of asthma in contrast to paracetamol (Lesko et al., 2002). In addition, investigations of the large databanks of Scandinavian and other countries indicate that paracetamol during pregnancy may:

  • (1) impair male fertility (Jensen et al., 2010; Snijder et al., 2012),

  • (2) hinder psychosocial development (Brandlistuen et al., 2013),

  • (3) increase the incidence of ADHD in later life (Liew et al., 2014).

• These observations have to be taken as signals of risk: Paracetamol use during pregnancy appears to interfere with the normal development of the newborn.

• They show less physical and psychological competence at the age of 3 years as compared to siblings from the same mother who had not been exposed to paracetamol during this pregnancy or were exposed to ibuprofen (Brandlistuen et al., 2013).

Cryptorchidism: Orchids are named after the Greek word for testicle, “orchis”. Cryptorchidism refers to an undescended testicle, the most prevalent congenital abnormality involving male genitalia.

• Moreover, preliminary evidence indicates that male children born from mothers taking paracetamol during pregnancy are more prone to suffer from cryptorchidism as compared to boys born without having been exposed to the drug (Jensen et al., 2010).

• The changes in the maturation of the immunological system of the fetus may cause an increased propensity to develop asthma, as immunological stem cells may not be programmed in an adequate manner (Thiele et al., 2013) and thus contribute to the increased incidence of asthma after the termination of aspirin use in children and pregnant women (Eder et al., 2006).

• Finally, the exposure of fertilized zebrafish eggs and larvae to NAPQI from paracetamol causes serious damage to the developing fish embryos (Weigt et al., 2010).

2.3 Glutathione Depletion

Time course of depletion

Slattery et al. (1987)12 gave 1 g of acetaminophen (APAP) to eight healthy adults (seven men and one woman), and studied the rates of excretion of APAP and its metabolites. Peak excretion of APAP occurred after 1.3 to 3.7 hours, and its mean half-life was 3.1 hours.

Another study used a higher dose, 3 g of APAP, and the renal clearance of acetaminophen and the formation clearances of the sulfate, glutathione, and catechol metabolites were lower by 38% and 35%, respectively, meaning that liver function was impaired.

In a third study (10 men), they found that 10 g of NAC reversed the liver depletion, increased the formation clearance of the sulphate conjugate by 27%, and glutathione conjugate by 10%.

In conclusion, they found that the hepatic supply of reduced glutathione and 3’-phosphoadenosine 5’-phosphosulfate begins to be depleted over the range of 0.5 to 3 g of APAP, and depletion is overcome by NAC.

Glutathione depletion and DNA damage

Sáez et al. (1993)13 incubated calf thymus DNA with two substances that promote the degradation of DNA: the antibiotic rifamycin SV and copper (II) ions. They found that glutathione helps to protect DNA from damage caused by rifamycin and copper-associated free radicals.

DNA damage rates are therefore likely to positively correlate with APAP dose and frequency of administration.

Dietary sources of phytonutrients for glutathione support

NAC is essential if overdose is suspected, but there are other reasons to optimise your glutathione status via diet; the most important of these is to reduce the risk of developing various chronic, age-related diseases.

Apart from protecting DNA, glutathione helps maintain our redox balance, reduces oxidative stress, enhances metabolic detoxification, and regulates immune system function.

Minich & Brown (2019)14 reviewed the literature and stressed the importance of maintaining a proper balance of glutathione, as too much may be harmful, for example, in cancer patients on chemotherapy.

Oral administration of glutathione is inefficient, with inconclusive findings from clinical studies. Relatively high doses are needed, and this is thought to be due to degradation by digestive peptidases. NAC supplementation gave similar, mixed results, but they suspect that it would work better synergistically, in combination with both cysteine and glycine.

4000 mg of omega-3 supplements taken daily for 12 weeks led to a better GSH–creatine ratio, with reduced symptoms of depression when compared to the control.

In another study, glutathione levels, antioxidant capacity, and inflammatory markers were all improved in Parkinson’s disease patients who’d been given 1000 mg of omega-3 for 12 weeks.

Other supplements and phytonutrients that were found to significantly support glutathione status included:

• B vitamins, including riboflavin, pantothenic acid (B5), and cobalamin (B12).

• Vitamin C, 500 or 1000 mg / day.

• Vitamin E (DL-alpha-tocopherol), 100 IU oral daily dose.

• Alpha-lipoic acid, 50 mg twice daily, or 300 mg three times daily.

• Selenium.

• Brassica vegetables (e.g. cabbage, broccoli, cauliflower, kale, and turnips).

• Allium vegetables (e.g. onions, garlic, leeks, shallots, and chives).

• Green tea.

• Fruit and vegetable juices (e.g. organic or conventional purple grape juice, Kale juice).

• Herbs and roots (e.g. rosemary, turmeric/curcumin, milk thistle, and Ginkgo biloba).

• Plant foods that contain glutathione (e.g. green foods, asparagus, avocado, cucumber, green beans, and spinach. See Tables 3 and 4).

2.4 Acetaminophen and immune response

Liver injury

Liu et al. (2004)15 used C57BL/6 lab mice to study the role of the immune system in APAP-induced liver injury. They found that natural killer (NK) and NK cells with T-cell receptors (NKT cells) played a critical role in the progression of liver injury by secreting interferon gamma (IFN-γ), by modulating chemokine production (inflammatory signalling leading to an accumulation of neutrophils), and by upregulating FasL expression in the liver. This further promotes an inflammatory response by the liver’s innate immune system.

They concluded that the toxic response to APAP is not just initiated by the highly reactive metabolite, N-acetyl-p-benzoquinone-imine (NAPQI), but that both NKT and NK cells play a critical role. Each NK cell type can compensate for the other to mediate APAP-induced liver injury.

Getachew et al. (2014)16 conducted further research using wild-type (C57BL/6J) and granzyme B-deficient (GrB -/-) mice. They concluded that APAP-induced liver injury was enhanced by the presence of activated, FasL expressing intrahepatic CD3 (+), CD4 (-), CD8 (-), NK1.1 (-) T cells.

Wang et al. (2015)17 also used male C57BL/6 mice (6–8 weeks old) and found that proinflammatory Th1 CD4+ T cells, but not CD8+ T cells modulated APAP-induced liver injury. Regulatory T cell (Trg) depletion augmented pro-inflammatory cytokines and exacerbated liver injury. Their investigations suggested that modulating the Th1/Treg balance may present a treatment option for APAP-induced liver injury.

Gamal et al. (2017)18 found that even low, therapeutic doses of APAP were associated with indicators of cellular stress with liver injury in the human liver:

Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier.

In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes.

In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury.

Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure.

… These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.

… TJ-disruption occurred in mouse liver at the lowest dose 75 mg/kg APAP, in the absence of classic indicators of drug-induced liver injury (liver ALT elevation). Given TJs are emerging as an important cellular signaling hub and may act as sensors for tissue stress and damage^5,27, our observations may prime future mechanistic studies to unravel complex TJ signaling pathways involved in APAP-induced stress.

… In agreement with Hu et al.⁴⁶, this pathology, observed at low-dose APAP in our study, may be particularly relevant in the context of potentiation of toxicity in ‘susceptibles’ with pre-existing liver disease/inflammatory stress, such as leaky TJs in NAFLD, or chronic low-dose ‘therapeutic’ APAP toxicity^45,46,47,48.

From: “Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver“ (2017)

https://www.nature.com/articles/srep37541

Acetaminophen and cancer

Najeebullah et al. published a short paper with the self-explanatory title: “Acetaminophen: A hazard to immunotherapy“ (2022).19

TLI5: If you are a cancer patient who is taking checkpoint inhibitors, then consider talking to your consultant about taking alternatives to APAP, as it stops them from working effectively.

If the exact mechanism of action is still a mystery, then how can you know it’s safe enough to be available on the shelves without a prescription?

• For a drug that is so widely used, the exact mechanism of action is still a mystery. However, different studies have led to the widespread belief that acetaminophen acts on the central serotonergic system to provide analgesic effects.

• Acetaminophen has been shown to penetrate the blood-brain barrier in humans, according to research [3]. As a result, it further strengthens the idea that APAP has a relationship to the serotonergic system.

• When taken as prescribed, acetaminophen is well tolerated; nevertheless, hypersensitivity, nephrotoxicity, and pancytopenia are some of the side effects of orally or rectally administered APAP.

• A recent study presented in the annual meeting of the American Society of Clinical Oncology and later on published in the Journal of Annals of Oncology in May 2022 raised new and more serious concerns — it revealed that cancer patients who take acetaminophen as premedication have a much-reduced response to immunotherapy.

• Immunotherapy works by blocking immune checkpoints, which enable immune cells to fight cancer more effectively [7].

• In a study, 600 patients with advanced cancer were given acetaminophen before starting immunotherapy, and the response of immune checkpoint inhibitors was assessed after the immunotherapy was started.

• The response of immune checkpoint inhibitors was dramatically reduced, implying that acetaminophen (APAP) has immunosuppressive effects [8].

• Acetaminophen (APAP) is frequently used to manage cancer-associated pain. A recent study looked at the effects of acetaminophen on cancer patients who were taking immune check inhibitors (ICB) [8].

• T cells can destroy tumor cells by blocking the binding of checkpoints which enable immune cells to fight cancer more effectively. T cells can destroy tumor cells by blocking the binding of checkpoint proteins like PDL1 to PD1 with immune checkpoint blockers [7].

• The effects of acetaminophen on a preclinical tumor model (MC 38) revealed that it significantly reduced the potency of ICB [9].

PD-1: “Programmed cell death protein 1(CD279 cluster of differentiation 279) is a protein encoded in humans by the PDCD1 gene.^[5][6] PD-1 is a cell surface receptor on T cells and B cells that has a role in regulating the immune system‘s response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells.^[7]”

• According to another study, acetaminophen therapy followed by interferon-gamma (20 ng/ml) for 2 h was linked to immune checkpoint PDL1 expression stimulation [10].

Infections are also promoted by APAP, and vaccines generate less of an immune response. This may not be such a bad thing, but perhaps take C, D, Zinc, etc. and avoid the need for vaccines and painkillers in the first place:

• Various other researchers have struggled to explain why acetaminophen suppresses the immune system. According to Neil et al. Aspirin and acetaminophen were tightly related to the reduction of the serum neutralizing antibody response towards the study challenge virus, Rhinovirus type 2.

Koufoglou et al. (2021)20 also found that:

“… The prophylactic administration of paracetamol decreased the immune response to certain pneumococcal serotypes in all included studies. The effect was more evident following primary vaccination and with immediate administration of paracetamol.”

“… The use of ibuprofen, that was evaluated in two studies, did not seem to affect the immunogenicity of PCVs .”

Ibuprofen is an alternative with fewer immunosuppressive properties, but it may double your risk of cardiovascular mortality when compared to aspirin alone. This is a typical finding for non-steroidal anti-inflammatory drugs (NSAIDs):

• Acetaminophen and aspirin both enhance RV2 viral shedding considerably. Ibuprofen had a weaker effect on antibody levels, and it did not differ significantly from placebo [11].

• Another identical research concluded that acetaminophen helps increase the duration of varicella infections in children, supporting the idea that acetaminophen reduces the human immune system [12]

• Paracetamol is given prophylactically before or shortly after the vaccination to reduce the pain and fever associated with vaccinations. However, new research has revealed that such prophylactic treatment may be interfering with the development of an immune response to vaccine antigens [13].

• When acetaminophen is administered prophylactically, it diminishes the response of Hepatitis B Antibodies (Anti-HBs) to Hepatitis B vaccination in adults.

The second (pointless) Hep B booster seemed to help, but antibody levels were still lower:

• According to a study Anti-HBs levels in the group receiving therapeutic paracetamol were found to be comparable to those in the control group a month after the second booster was administered. However, Anti HBs levels were noticeably lower in. the paracetamol group is given as a preventive [14].

• It was also revealed from Animal trials that acetaminophen (APAP) decreases the antibody response towards Sheep red blood cells (SRBC) in both fed and fasted mice indicating acetaminophen has an immunosuppressive impact on humoral immunity [15].

An editorial also discussed primary research by Bessede et al.:

Cited paper from 2022:

Impact of acetaminophen on the efficacy of immunotherapy in cancer patients

Highlights

•APAP has been shown to blunt the antibody response to vaccination.

•APAP may impact the efficacy of immunotherapy in patients with cancer.

•Further research is needed to decipher the impact of APAP on immunity.

Abstract

Background

Acetaminophen (APAP) use has been associated with blunted vaccine immune responses. This study aimed to assess APAP impact on immunotherapy efficacy in patients with cancer.

Patients and methods

Exposure to APAP was assessed by plasma analysis and was correlated with clinical outcome in three independent cohorts of patients with advanced cancer who were treated with immune checkpoint blockers (ICBs). The immunomodulatory effects of APAP were evaluated on a preclinical tumor model and on human peripheral blood mononuclear cells (PBMCs) from healthy donors.

Results

Detectable plasma APAP levels at treatment onset were associated with a significantly worse clinical outcome in ICB-treated cancer patients, independently of other prognostic factors. APAP significantly reduced ICB efficacy in the preclinical MC38 model, as well as the production of PD-1 blockade-related interferon-γ secretion by human PBMCs.

Tregs may help limit APAP-induced liver damage, but they also promote tumour growth by suppressing the immune system:

Moreover, reduction of ICB efficacy in vivo was associated with significantly increased tumor infiltration by regulatory T cells (Tregs). Administration of APAP over 24 h induced a significant expansion of peripheral Tregs in healthy individuals.

In addition, interleukin-10, a crucial mediator of Treg-induced immune suppression, was significantly up-regulated upon treatment with ICB in cancer patients taking APAP.

Conclusions

This study provides strong preclinical and clinical evidence of the role of APAP as a potential suppressor of antitumor immunity. Hence, APAP should be used with caution in patients treated with ICB.

Overall and progression-free survival of cancer patients is on the Y-axis of B, C and D. APAP cohorts are denoted by the brown curve.

Did your doctor or pharmacist tell you this?

More: https://www.annalsofoncology.org/article/S0923-7534(22)01208-X/fulltext

Now, where did I put the portable Milwaukee drug disposal toilet?

The following year, Nelli et al. published follow-up research, but this time the cohort was a group of patients with a type of advanced lung cancer associated with very low 5-year survival rates of around 7%. APAP lowers this further still!

In stage 4 NSCLC, the cancer has become metastatic and has spread from its original location to distant parts of the body.

They mention corticosteroids, and I reviewed the literature in a Substack, discussing how they are associated with a worse prognosis. This was partly due to inhibition of the key tumour suppressor p53:

Effects of Acetaminophen Exposure on Outcomes of Patients Receiving Immune Checkpoint Inhibitors for Advanced Non-Small-Cell Lung Cancer: A Propensity Score-Matched Analysis

Abstract

(1) Background:

Several studies have investigated potential interactions between immune checkpoint inhibitors (ICIs) and commonly prescribed medications. Although acetaminophen (APAP) has not been considered susceptible to interaction with ICIs, recent research has shown that detectable plasma levels of this drug can hinder the efficacy of PD-1/PD-L1 blockade therapies.

A reliable assessment of the potential interaction between APAP and ICIs in advanced non-small cell lung cancer (NSCLC) patients would be worthwhile since it is often prescribed in this condition.

We sought to evaluate the impact of the concomitant use of APAP in patients with advanced NSCLC on PD-1/PD-L1 blockade using real-world evidence.

The patients were on courses of chemo- or immunotherapy:

(2) Methods:

This study included consecutive patients with histologically proven stage IV NSCLC who underwent first-line therapy with pembrolizumab as a single agent or in combination with platinum-based chemotherapy, or second-line therapy with pembrolizumab, nivolumab, or atezolizumab.

The intensity of APAP exposure was classified as low (therapeutic intake lasting less than 24 h or a cumulative intake lower than 60 doses of 1000 mg) or high (therapeutic intake lasting more than 24 h or a total intake exceeding 60 doses of 1000 mg).

The favorable outcome of anti-PD-1/PD-L1 therapies was defined by durable clinical benefit (DCB). Progression-free survival (PFS) and overall survival (OS) were relevant to our efficacy analysis. Propensity score matching (PSM) methods were applied to adjust for differences between the APAP exposure subgroups.

(3) Results:

Over the course of April 2018 to October 2022, 80 patients were treated with first-line pembrolizumab either as single-agent therapy or in combination with platinum-based chemotherapy.

During the period from June 2015 to November 2022, 145 patients were given anti-PD-1/PD-L1 blockade therapy as second-line treatment. Subsequent efficacy analyses relied on adjusted PSM populations in both treatment settings.

Multivariate testing revealed that only the level of APAP and corticosteroid intake had an independent effect on DCB in both treatment lines. Multivariate Cox regression analysis confirmed high exposure to APAP and immunosuppressive corticosteroid therapy as independent predictors of shorter PFS and OS in both treatment settings.

(4) Conclusions:

Our findings would strengthen the available evidence that concomitant intake of APAP blunts the efficacy of ICIs in patients with advanced NSCLC. The detrimental effects appear to depend on the cumulative dose and duration of exposure to APAP. The inherent shortcomings of the current research warrant confirmation in larger independent series.

“Lines of therapy is a term that we use to describe the order in which different treatments are given to people as their cancer progresses,” explained Jyoti Patel, MD, medical director of thoracic oncology and assistant director for clinical research at the Lurie Cancer Center of Northwestern University, in Chicago.

A first-line therapy is simply the first cancer treatment you receive. If your cancer progresses, or if the toxicities (side effects) of treatment become intolerable, a doctor may suggest a second-line treatment. The next treatment after that would be third-line, and so on.”21

First-line APAP vs steroids. As before, the Y-axis denotes % survival:

Second-line APAP vs steroids.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC10527930/

2.5 Acetaminophen and harm to the child

I’ve selected 10 papers for review, from 1982. Please follow the links for further reading. They could all justify having their own Substack.

This amount of research should have raised alarm bells with the regulators decades ago, or class action claims as seen with thalidomide and valproate.

As ever, I hope this Substack helps you to have informed consent and to be your own drug regulator, the un-captured kind. These sorts of toxic substances have no place on a supermarket shelf. They should be locked away in medicine cabinets.

From Wiki:

“Teratogenesis occurs when the development of an embryo is altered negatively due to the presence of teratogens. Teratogens are the causes of teratogenesis. Common examples of teratogens include genetic disorders, maternal nutrition and health, and chemical agents such as drugs and alcohol.[10]

Lesser known examples that will be covered include stress,[11] caffeine,[12] and deficiencies in diet and nutrition.[13]

Although teratogens can affect a fetus during any time in the pregnancy, one of the most sensitive time frames for them to be exposed to the developing embryo is during the embryonic period.

This period is in effect from about the fourteenth day following when a female’s egg is implanted into a specific place in the reproductive organs and sixty days after conception.[14] Teratogens are able to cause abnormal defects through certain mechanisms that occur throughout the development of the embryo.”

The pharma-sponsored NHS is on top form as ever, complete with in-your-face lies:

From 1982 (paywalled), by Golden et al.:

Propoxyphene and acetaminophen. Possible effects on the fetus

Abstract

Propoxyphene (Darvon) and acetaminophen (Tylenol) are widely prescribed analgesic agents. Both can cross the placenta, and propoxyphene can produce serious withdrawal symptoms in newborns. Neither propoxyphene nor acetaminophen is considered a teratogen, yet, three malformed infants who were antenatally exposed to propoxyphene have previously been reported.

We report a fourth case of an infant, with withdrawal symptoms and cranial-facial and digital malformations, born to a women who used propoxyphene and acetaminophen throughout her pregnancy. We suggest that possibility that the antepartum use of propoxyphene and acetaminophen, in combination, may be teratogenic.

More: https://pubmed.ncbi.nlm.nih.gov/6890421/

Aspirin, also an NSAID, has strong anti-cancer properties and cardiovascular benefits, but is also associated with harm to the exposed child.

From 1987, by Streissguth et al.:

Aspirin and acetaminophen use by pregnant women and subsequent child IQ and attention decrements

Abstract

In a longitudinal prospective study of 1,529 women pregnant in 1974–1975, aspirin and acetaminophen were the two medications most frequently taken during the first half of pregnancy (46 and 41%, respectively).

In a selected cohort of 421 offspring of these women, examined at 4 years of age, maternal aspirin use during the first half of pregnancy was significantly related to IQ and attention decrements in the exposed children.

Multiple regression analyses were used to statistically adjust for a variety of potentially confounding factors including demographic characteristics, child characteristics, other exposures, and lifestyle/environmental variables.

Continuous doseresponse and step-function parameterizations of aspirin exposure were both statistically significant and not clearly distinguishable from each other.

The estimated aspirin effect is significantly greater, for girls than boys. Aspirin effects on offspring function were found in the absence of effects on physical size both at birth and at 4 years.

In this study, the effect of APAP alone was not significant:

Maternal acetaminophen use was not significantly related to child IQ or attention. As this exploratory research originated from observations of a data set gathered for other purposes, it would be desirable to have these findings replicated in other studies. Further follow-up of the children at a later age is planned.

More (paywalled): https://onlinelibrary.wiley.com/doi/abs/10.1002/tera.1420350207

In 2017, Culha & Serefoglu discussed in a brief editorial how APAP can have a negative impact on male fertility. They called for more research and for policymakers to take these findings into consideration.

Reproduced in full:

Prolonged exposure to acetaminophen during pregnancy reduces testosterone production by the human fetal testis

Congenital anomalies are a major cause of infant morbidity and mortality, and a significant proportion of these anomalies involve urogenital malformations (1). As fetal testosterone has crucial importance in the development of the male reproductive system, any disruption in its production can result in disorders such as hypospadias, cryptorchidism or testicular cancer (2).

The risk of congenital malformation, including urogenital abnormalities, is increased by the administration of medical treatments during pregnancy (3). Weak analgesics, particularly acetaminophen containing over-the-counter drugs, are widely used among pregnant women in Europe and North America (4).

However, recent studies have demonstrated that these analgesics may impair fetal testicular hormone production (5,6) and increase the risk of cryptorchidism in the offspring (7). Moreover, some have reported that this risk is even further promoted by the combination of acetaminophen-containing medications with other analgesics (5).

In an elegant study, van den Driesche et al. (8) demonstrated that protracted exposure of xenografted human testes to a therapeutic dose of acetaminophen decreased the plasma levels of testosterone, as well as decreased the weight of seminal vesicles in castrated host mice.

After recording a significant reduction in both Cyp11a1 and Cyp17a1 mRNA expression after the final dose of acetaminophen administration via reverse transcription polymerase chain reaction, the authors concluded that the acetaminophen-induced testosterone reduction may result from the reduced quantities of these key steroidogenic enzymes (8).

With these important findings, the study of van den Driesche et al. (8) sheds light to the unknown aspects of the impact of endocrine disrupting compounds on fetal testosterone production.

The detrimental effect of widely used acetaminophen containing analgesics on testosterone production must be taken into consideration by policy makers in order to heighten public awareness regarding the potential teratogenic effects of these over-the-counter analgesics. Meanwhile, researchers should corroborate the findings of this study in order to develop further preventive measures, if necessary.

From: https://pmc.ncbi.nlm.nih.gov/articles/PMC5451610/

Bearing in mind that child vaccination schedules present confounding factors, this systematic review from 2018 by Masarwa et al. still found significant clinical signals for ADHD, ASD, and hyperactivity:

Prenatal Exposure to Acetaminophen and Risk for Attention Deficit Hyperactivity Disorder and Autistic Spectrum Disorder: A Systematic Review, Meta-Analysis, and Meta-Regression Analysis of Cohort Studies

… Seven eligible retrospective cohorts included 132,738 mother-child pairs, with follow-up periods ranging from 3 to 11 years.

The pooled risk ratio for ADHD was 1.34 (95% confidence interval (CI): 1.21, 1.47; I² = 72%); for ASD, the risk ratio was 1.19 (95% CI: 1.14, 1.25; I² = 14%), and for hyperactivity symptoms, it was 1.24 (95% CI: 1.04, 1.43; I² = 93%).

In meta-regression analysis, the association between exposure and ADHD increased with the child’s age upon follow-up (β = 0.03, 95% CI: 0.00, 0.07) and with the mean duration of exposure (β = 0.00, 95% CI: 0.00, 0.01).

The available data is of observational nature only. Studies differed widely in exposure and outcome assessment.

Acetaminophen use during pregnancy is associated with an increased risk for ADHD, ASD, and hyperactivity symptoms.

These findings are concerning; however, results should be interpreted with caution given that the available evidence consists of observational studies and is susceptible to several potential sources of bias.

More: https://academic.oup.com/aje/article-abstract/187/8/1817/4980325

A further study, from 2019 by Ji et al., analysed the relationship between cord plasma APAP biomarker levels and ADHD/hyperactivity outcomes and also found a significantly increased risk—up to 4.1 fold higher.

This differs from previous research in that it didn’t rely on maternal self-report.

Acetaminophen glucuronide: an inactive, major metabolite of APAP that is formed in the liver and primarily excreted in the urine.

3-[N-acetyl-l-cystein-S-yl]-acetaminophen: a metabolite of acetaminophen and a biomarker for liver injury caused by APAP.

Association of Cord Plasma Biomarkers of In Utero Acetaminophen Exposure With Risk of Attention-Deficit/Hyperactivity Disorder and Autism Spectrum Disorder in Childhood

Key Points

Question What is the association between cord plasma biomarkers of in utero acetaminophen exposure and risk of childhood attention-deficit/hyperactivity disorder and autism spectrum disorder?

Findings In this cohort study of 996 mother-infant dyads from the Boston Birth Cohort, cord plasma biomarkers of fetal exposure to acetaminophen were associated with significantly increased risk of childhood attention-deficit/hyperactivity disorder and autism spectrum disorder.

Meaning These findings suggest in utero exposure to acetaminophen is associated with increased risk of attention-deficit/hyperactivity disorder and autism spectrum disorder in children and warrant additional investigations.

Abstract

Importance Prior studies have raised concern about maternal acetaminophen use during pregnancy and increased risk of attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) in their children; however, most studies have relied on maternal self-report.

Objective To examine the prospective associations between cord plasma acetaminophen metabolites and physician-diagnosed ADHD, ASD, both ADHD and ASD, and developmental disabilities (DDs) in childhood.

Design, Setting, and Participants This prospective cohort study analyzed 996 mother-infant dyads, a subset of the Boston Birth Cohort, who were enrolled at birth and followed up prospectively at the Boston Medical Center from October 1, 1998, to June 30, 2018.

Exposures Three cord acetaminophen metabolites (unchanged acetaminophen, acetaminophen glucuronide, and 3-[N-acetyl-l-cystein-S-yl]-acetaminophen) were measured in archived cord plasma samples collected at birth.

Main Outcomes and Measures Physician-diagnosed ADHD, ASD, and other DDs as documented in the child’s medical records.

Results Of 996 participants (mean [SD] age, 9.8 [3.9] years; 548 [55.0%] male), the final sample included 257 children (25.8%) with ADHD only, 66 (6.6%) with ASD only, 42 (4.2%) with both ADHD and ASD, 304 (30.5%) with other DDs, and 327 (32.8%) who were neurotypical.

Unchanged acetaminophen levels were detectable in all cord plasma samples. Compared with being in the first tertile, being in the second and third tertiles of cord acetaminophen burden was associated with higher odds of ADHD diagnosis (odds ratio [OR] for second tertile, 2.26; 95% CI, 1.40-3.69; OR for third tertile, 2.86; 95% CI, 1.77-4.67) and ASD diagnosis (OR for second tertile, 2.14; 95% CI, 0.93-5.13; OR for third tertile, 3.62; 95% CI, 1.62-8.60).

Sensitivity analyses and subgroup analyses found consistent associations between acetaminophen buden and ADHD and acetaminophen burden and ASD across strata of potential confounders, including maternal indication, substance use, preterm birth, and child age and sex, for which point estimates for the ORs vary from 2.3 to 3.5 for ADHD and 1.6 to 4.1 for ASD.

Conclusions and Relevance Cord biomarkers of fetal exposure to acetaminophen were associated with significantly increased risk of childhood ADHD and ASD in a dose-response fashion. Our findings support previous studies regarding the association between prenatal and perinatal acetaminophen exposure and childhood neurodevelopmental risk and warrant additional investigations.

Vaccination status is missing, but is likely to apply to over 90% of these subgroups, according to recent UK data:22

More: https://jamanetwork.com/journals/jamapsychiatry/fullarticle/2753512

N.B. What’s the story with “bottle only” feeding? It’s not the focus of this SubStack, but other research from 2023 found that breast is best:

In 2022, a paper by Rosas-Ramirez et al. managed to get published, even though it had the T-word in the title.

We learned earlier how NAPQI binds with hepatic mitochondrial proteins, disrupts the electron transport chain (ETC) and that this leads to peroxide formation and ROS generation. This paper links that oxidation with teratogenic outcomes:

Teratogenic effects induced by paracetamol, ciprofloxacin, and their mixture on Danio rerio embryos: Oxidative stress implications

Highlights

• Developmental abnormalities and oxidative stress were evaluated in zebrafish.

• Paracetamol, ciprofloxacin and their mixtures can alter fish embryogenesis.

• Paracetamol, ciprofloxacin and their mixture induced oxidative damage in fish embryos.

• Concentrations of evaluated drugs and their mixtures pose a threat to aquatic species.

Abstract

Even though the toxic effects of paracetamol (PCM) and ciprofloxacin (CPX) have been deeply studied in the last decades, the impact of the PCM-CPX mixture may induce in aquatic organisms is poorly known. Thus, the objective of this work was to investigate the teratogenic effects and oxidative stress that PCM, CPX, and their mixture induce in Danio rerio embryos.

Moreover, we aimed to determine whether the PCM-CPX mixture induces more severe effects on the embryos than the individual drugs. For this purpose, zebrafish embryos (4 hpf) were exposed to environmentally relevant concentrations of PCM, CPX, and their mixture until 96 hpf.

In addition, at 72 hpf and 96 hpf, we also evaluated the oxidative stress biomarkers (superoxide dismutase, catalase, glutathione peroxidase, lipid peroxidation, and hydroperoxides and carbonyl content) in the embryos.

Our results demonstrated that PCM, CPX, and their mixture reduced the survival rate of embryos by up to 75%. In addition, both drugs, induced morphological alterations in the embryos, causing their death.

The most observed malformations were: scoliosis, craniofacial malformations, hypopigmentation, growth retardation, pericardial edema.

Concerning oxidative stress, our integrated biomarkers response (IBR) analysis demonstrated that PCM, CPX, and their mixture induce oxidative damage on the embryos.

In conclusion, PCM, CPX, and their mixture can alter zebrafish embryonic development via an oxidative stress response.

This also has implications for the health of the aquatic environment.

For further reading, see:

“Pharmaceuticals: A Headache for Water Treatment” (2006)

“Occurrence of Paracetamol in Aquatic Environments and Transformation by Microorganisms: A Review” (2017)

According to Acetaminophen Global Market Report, 2018, worldwide sales of PCM in 2019 summed a total of 740 million USD. Nonetheless, it is forecasted that by 2023 its sales will rise to 780 million USD.

As a result of its high usage and partial degradation (14–75%), huge amounts of PCM are spilled out every day into the aquatic environment (Gutiérrez-Noya et al., 2021). Recent studies have reported the occurrence of this drug in worldwide water bodies.

Fig. 1 shows the proportions of live, dead, and malformed embryos for each concentration of PCM, CPX, and their binary mixtures. In all cases, the number of live embryos decreased with increasing drug concentration.

Ciprofloxacin had lower death rates. However, the PCM-CPX mixtures showed the highest rates of deaths and malformations compared to the individual drugs. The M4 mixture showed lower numbers of live embryos.

Fig. 3 shows the effect-concentration curves of PCM, CPX and the four binary mixtures PCM-CPX tested about the normal development of zebrafish embryos according to the score of Hermsen et al., 2011.

In all cases, a decrease in the score is shown with respect to the control organisms, due to the different morphological alterations presented. Concentrations from 300 to 1200 μg L⁻¹ of PCM showed statistically significant differences in relation to the control group (p < 0.05). In the case of CPX, the concentration that showed the greatest influence on embryo development was 25 μg L⁻¹.

Meanwhile, the concentrations from 15 μg L⁻¹ showed significant differences with respect to the control group (p < 0.05). In the case of binary mixtures of PCM-CPX, M4 was the only mixture that showed significant differences with respect to the control group.

More: https://www.sciencedirect.com/science/article/abs/pii/S0048969721056187

Klein et al. (2023) investigated the effects of gestational APAP on rats. Their research helped to support the hypothesis that APAP leads to endocannabinoid (eCB) dysfunction, and this harms the developing immune system, leading to hyperactivity, ASD, and ADHD:

Gestational paracetamol exposure induces core behaviors of neurodevelopmental disorders in infant rats and modifies response to a cannabinoid agonist in females

Highlights

• Prenatal exposure to paracetamol increases motor activity and repetitive behavior in male and female rats.

• Increased apomorphine-induced stereotypies and reduced anxiety were observed in paracetamol-exposed females.

• Prenatal paracetamol modified the response to a cannabinoid agonist in exposed females.

• Results suggest a role for paracetamol in affecting neurodevelopment.

• Paracetamol may harm the developing brain by inducing endocannabinoid dysfunction.

Abstract

Paracetamol (PAR) is an over-the-counter analgesic/antipyretic used during pregnancy worldwide. Epidemiological studies have been associating gestational PAR exposure with neurobehavioral alterations in the progeny resembling autism spectrum disorders and attention-deficit hyperactivity disorder symptoms.

The endocannabinoid (eCB) dysfunction was previously hypothesized as one of the modes of action by which PAR may harm the developing nervous system. We aimed to evaluate possible effects of gestational exposure to PAR on male and female rat’s offspring behavior and if an acute injection of WIN 55,212–2 (WIN, 0.3 mg/kg), a non-specific cannabinoid agonist, prior to behavioral tests, would induce different effects in PAR exposed and non-exposed animals.

Pregnant Wistar rats were gavaged with PAR (350 mg/kg/day) or water from gestational day 6 until delivery. Nest-seeking, open field, apomorphine-induced stereotypy, marble burying and three-chamber tests were conducted in 10-, 24-, 25- or 30-days-old rats, respectively.

PAR exposure resulted in increased apomorphine-induced stereotyped behavior and time spent in the central area of the open field in exposed female pups.

Additionally, it induced hyperactivity in the open field and increased marble burying behavior in both male and female pups. WIN injection modified the behavioral response only in the nest seeking test, and opposite effects were observed in control and PAR-exposed neonate females.

Reported alterations are relevant for the neurodevelopmental disorders that have been associated with maternal PAR exposure and suggest that eCB dysfunction may play a role in the action by which PAR may harm the developing brain.

… Approximately 40–50% of pregnant women use PAR at least once during gestation worldwide (Bandoli et al., 2019; Rifas-Shiman et al., 2020; Sznajder et al., 2022) and part of them repeat the use for weeks during pregnancy (Bandoli et al., 2019).

Considering PAR and some of its metabolites cross the placental barrier (Conings et al., 2019; Nitsche et al., 2017) there is fetal exposure as a result of maternal intake of this medicine.

PAR exposure increased latency to reach nest bedding in comparison to CTL group. However, WIN injection reversed the response profiles, reducing latency values for PAR-exposed females and increasing for CTL group (Fig. 2).

Hyperactive behaviour presented as more time spent in the central area of the open field:

Average time in central area was 0.63 standard deviations higher than in CTL females (Cohen’s d = 0.63). Therefore, independently of WIN injection, PAR-exposed females spent more time in the central area of the open field. No other effects of exposure, sex, WIN injection or interaction among them were observed (Supplementary Table 3).

Apomorphine: “… a type of Parkinson’s drug. It comes as a liquid. It is part of the class (category) of drugs called dopamine agonists.

Dopamine agonists work by tricking your brain into thinking they are dopamine. This means they can copy the way dopamine works and can reduce your symptoms.

Apomorphine does not have morphine in it.”23

The split of the data into sex followed by two-way ANOVAs indicated that the exposure effect was significant only in females (F_{(1; 66)} = 17.808, p = 0.000) but not in males (F_{(1; 58)} = 0.618, p = 0.435) (Fig. 4). Exposure factor in females accounted for 21% of data variability (η_p² = 0.21, 90% CI [0.08, 0.34]) and average stereotypy intensity of PAR-exposed females was 1.04 standard deviations greater than CTL females (Cohen’s d = 1.04). In this way, independently of injection, PAR exposure resulted in increased stereotypy intensity in female pups.

APAP (PAR)-associated repetitive behaviour:

More (paywalled): https://pubmed.ncbi.nlm.nih.gov/37391024/

A Chinese retrospective study of pregnant women, from 2024, by Xu et al. is alarming. We know that zebrafish embryo mortality increased with dose and duration of exposure. This study found similar effects on the stillbirth rate.

Vaccine status wasn’t defined or discussed, but the compliance rate was likely to have been very high. It would be useful for research to be funded which investigates the synergistic effects of APAP exposure and vaccination.

Apgar score: “The Apgar score is a standardized assessment of a neonate’s status immediately after birth and the response to resuscitation efforts and remains the gold standard for evaluating neonates.”

“There are 5 parts to an Apgar score. Each category is weighted evenly and assigned a 0, 1, or 2 value. The components are then added to give a score recorded 1 and 5 minutes after birth.

A score of 7 to 10 is considered reassuring, a score of 4 to 6 is moderately abnormal, and a score of 0 to 3 is deemed low in full-term and late preterm infants, at 5 minutes, when an infant has a score of <7, Neonatal Resuscitation Program guidelines recommend continued recording at 5-minute intervals up to 20 minutes.”24

They found that acetaminophen use more than doubled the risk of stillbirth and Apgar scoring below 7.

Association of acetaminophen use with perinatal outcomes among pregnant women: a retrospective cohort study with propensity score matching

Abstract

Background

Although acetaminophen is widely used in women during pregnancy, its safety has not been clearly stated. The study aimed to investigate the association between acetaminophen use and adverse pregnancy outcomes in pregnant women in China.

Methods

We conducted a retrospective cohort study by collecting data on pregnant women who delivered in the Beijing Obstetrics and Gynecology Hospital from January 2018 to September 2023. An acetaminophen use group and a control group were formed based on prenatal exposure to acetaminophen.

The pregnancy outcomes that we focused on were stillbirth, miscarriage, preterm birth, APGAR score, birth weight, and congenital disabilities. Pregnant women exposed to acetaminophen were matched to unexposed in a 1:1 ratio with propensity score matching, using the greedy matching macro. SPSS software was used for statistical analysis. Multivariable logistics regression was used to assess the association between acetaminophen use during pregnancy and adverse pregnancy outcomes.

Results

A total of 41,440 pregnant women were included, of whom 501 were exposed to acetaminophen during pregnancy, and 40,939 were not exposed. After the propensity score matching, the acetaminophen use and control groups consisted of 501 pregnant women each.

The primary analysis showed that acetaminophen exposure during pregnancy was associated with an increased risk of stillbirth (adjusted OR (aOR) = 2.29, 95% CI, 1.19–4.43), APGAR score < 7 at 1 min (aOR = 3.28, 95% CI, 1.73–6.21), APGAR score < 7 at 5 min (aOR = 3.54, 95% CI, 1.74–7.20), APGAR score < 7 at 10 min (aOR = 3.18, 95% CI, 1.58–6.41), and high birth weight (HBW) (aOR = 1.75, 95% CI, 1.05–2.92).

Drug exposure during the first and second trimesters increased the odds of stillbirth, miscarriage, APGAR < 7, and the occurrence of at least one adverse pregnancy outcome. In addition, the frequency of drug use more than two times was associated with a higher risk of preterm birth and APGAR score < 7.

Conclusions

Exposure to acetaminophen during pregnancy was significantly associated with the occurrence of adverse pregnancy outcomes, particularly exposure in the first and second trimesters and frequency of use more than twice. It is suggested that acetaminophen should be prescribed with caution in pregnant women.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC11010302/

What do you say to that, NHS?

Note: Of the 41,440 pregnant women, only 501 were confirmed to have been exposed to APAP, and 40,939 were not exposed. If its use is so widespread, then why was the take-up so low?

Even the FDA now advises caution:

If you thought that APAP-associated toxicity in fetal offspring couldn’t get any worse, you’d be wrong.

A Chinese study from 2024 by Chen et al. used male and female mice and found Prenatal acetaminophen exposure (PAcE)-induced multi-organ developmental toxicity:

The characterization of developmental toxicity in fetal offspring induced by acetaminophen exposure during pregnancy

Highlights

• Prenatal acetaminophen exposure (PAcE) altered male and female fetus blood phenotypes.

• Functional genes in multiple organs are affected by PAcE.

• Significant alterations manifest after high-dose, second-trimester, and multi-course PAcE

• Male fetuses are more affected by PAcE than females.

Abstract

Objective

Acetaminophen (APAP), an antipyretic and analgesic commonly used during pregnancy, has been recognized as a novel environmental contaminant. Preliminary evidence suggests that prenatal acetaminophen exposure (PAcE) could adversely affect offspring’s gonadal and neurologic development, but there is no systematic investigation on the characteristics of APAP’s fetal developmental toxicity.

Methods

Pregnant mice were treated with 100 or 400 mg/kg∙d APAP in the second-trimester, or 400 mg/kg∙d APAP in the second- or third-trimester, or different courses (single or multiple) of APAP, based on clinical regimen.

The effects of PAcE on pregnancy outcomes, maternal/fetal blood phenotypes, and multi-organ morphological and functional development of fetal mice were analyzed.

Results

PAcE increased the incidence of adverse pregnancy outcomes and altered blood phenotypes including aminotransferases, lipids, and sex hormones in dams and fetuses.

The expression of key functional genes in fetal organs indicated that PAcE inhibited hippocampal synaptic development, sex hormone synthesis, and osteogenic and chondrogenic development, but enhanced hepatic lipid synthesis and uptake, renal inflammatory hyperplasia, and adrenal steroid hormone synthesis.

PAcE also induced marked pathological alterations in the fetal hippocampus, bone, kidney, and cartilage.

The sensitivity rankings of fetal organs to PAcE might be hippocampus/bone > kidney > cartilage > liver > gonad > adrenal gland. Notably, PAcE-induced multi-organ developmental toxicity was more considerable under high-dose, second-trimester, and multi-course exposure and in male fetuses.

Conclusion

This study confirmed PAcE-induced alterations in multi-organ development and function in fetal mice and elucidated its characteristics, which deepens the comprehensive understanding of APAP’s developmental toxicity.

However, the safety of APAP during pregnancy is not entirely assured (de Castro et al., 2022, Feldkamp et al., 2010). Numerous investigations on birth cohorts have indicated that prenatal acetaminophen exposure (PAcE) may result in negative outcomes during pregnancy, like premature birth and low birth weight in newborns (Bandoli et al., 2020).

Furthermore, PAcE also exerts an influence on the health status of offspring after birth: follow-up of a Danish birth cohort from 1996 to 2002 found that PAcE was linked to an increased likelihood of obesity in female children (Liew et al., 2019); a Norwegian cohort research exhibited a significant correlation between PAcE and attention-deficit and hyperactivity disorder (ADHD) in children (Ystrom et al., 2017); another cohort study in Boston, USA, which included 996 mother-child pairs, showed that there is a correlation between PAcE and a higher likelihood of asthma and allergic rhinitis in the children (Sordillo et al., 2019); a UK cohort study that enrolled pregnant women who delivered infants in 2001–2009 and followed their babies to 24 months of age found that PAcE during the gestational week 8–14 was associated with shorter anogenital spacing in male children (Fisher et al., 2016). Therefore, the necessity of evaluating the safety of PAcE and its effects on the development of the offspring should be emphasized.

Apart from that, it’s quite safe.

Our prior study has shown that when the mother is exposed to drugs, it can cause harmful effects on the development of the baby and lead to malfunction in several organs (Lu et al., 2023b).

Research shows that PAcE may cause abnormalities in the development of the offspring’s neurological, reproductive, and urogenital systems (Konkel, 2018, Kristensen et al., 2016): PAcE may cause autism spectrum disorder (ASD) (Ji et al., 2020) as well as testicular dysplasia in the offspring (Fisher et al., 2016).

My review of HPV vaccine toxicology was prompted by investigating the links to osteoporosis. Aluminium adjuvants suppress the Wnt/β-catenin signalling pathway. APAP also suppresses this by decreasing the expression of a gene that codes for alpha-1 type I collagen.

In this study, PAcE caused neurodevelopmental deficits in the male fetal hippocampus, which was consistent with pathological findings. This may be linked to the reduction of glutathione (GSH) levels and striatal superoxide dismutase (SOD) activity in the hippocampus of male offspring by PAcE (Rigobello et al., 2021).

The Wnt signaling system has been found to exert a positive regulatory influence on Col1a1 in bones (Zhang et al., 2018). PAcE decreases the expression of Col1a1 by suppressing the activity of the Wnt signaling pathway, resulting in fetal bone hypoplasia (Ma et al., 2023).

In this study, Von Kossa staining, which reflects bone mineralization, also showed that the bone trabecula was thinner in both male and female fetuses in the ACH group.

This study also found that there were glomerular vacuoles in the fetal kidneys of the ACH group, suggesting fetal glomerular dysplasia, but its underlying mechanism needs to be further studied.

In cartilage, Col2a1 is positively regulated by transforming growth factor β (TGFβ), and PAcE can inhibit the activity of the TGFβ pathway, leading to decreased expression of Col2a1, which may result in fetal chondrodysplasia (Zhang et al., 2023).

Furthermore, PAcE hinders the growth of spermatogonial cells and stimulates cell death by activating the Wnt pathway and inhibiting the Notch signaling pathway, which ultimately changes the typical course of fetal testicular development (Liu et al., 2023). In general, the severity of morphological changes in different organs is consistent with the organ sensitivity rankings.

Having described teratogenic effects throughout, they added this line to section 4.2, presumably to get past the censor:

“In the present study, we did not observe an obvious teratogenic effect of PAcE under clinical doses, which is consistent with the conclusions of a clinical case from the REACH registration dossier (Lederman et al., 1983) and an early animal study (Lambert and Thorgeirsson, 1976).”

Whereas in their conclusion, they directly contradict themselves:

5. Conclusion

By establishing PAcE mouse models that mimicked the clinical drug regimens, this study indicated that PAcE affects pregnancy outcomes, maternal/fetal blood phenotypes, and multi-organ morphological and functional development of fetal mice.

These effects had dose, stage, course, and sex differences, with the most significant changes under high-dose, second-trimester, and multi-course exposure and in male fetuses.

These findings also suggest that pregnant women should avoid APAP or use APAP cautiously in the short term at the lowest effective dose. In conclusion, this study comprehensively evaluates the fetal developmental toxicity of APAP, deepens our understanding of the health risks of PAcE, and establishes a crucial theoretical and experimental foundation for further evaluating the long-term harm of PAcE and its programming mechanisms.

More: https://www.sciencedirect.com/science/article/pii/S014765132401056X

Our final paper was a systematic review by Prada et al., which was published earlier this year:

Evaluation of the evidence on acetaminophen use and neurodevelopmental disorders using the Navigation Guide methodology

… Results

We identified 46 studies for inclusion in our analysis. Of these, 27 studies reported positive associations (significant links to NDDs), 9 showed null associations (no significant link), and 4 indicated negative associations (protective effects). Higher-quality studies were more likely to show positive associations.

Overall, the majority of the studies reported positive associations of prenatal acetaminophen use with ADHD, ASD, or NDDs in offspring, with risk-of-bias and strength-of-evidence ratings informing the overall synthesis.

Conclusions

Our analyses using the Navigation Guide thus support evidence consistent with an association between acetaminophen exposure during pregnancy and increased incidence of NDDs.

Appropriate and immediate steps should be taken to advise pregnant women to limit acetaminophen consumption to protect their offspring’s neurodevelopment.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC12351903/

I have to concur, and the NHS should also fall into line as a matter of urgency.

3.0 Parting shots

3.1 Flu vax failure… again

That naughty virus went ahead and mutated.

They gambled wrongly on which strain to target. Although the vax doesn’t work, the fix is always the same:

Flu spreading ‘unusually early’ in Europe amid new strain, health officials warn

Health officials are bracing for a harsh flu season in Europe as a new viral strain spreads “unusually early” across the bloc.

Authorities are detecting influenza cases three to four weeks earlier than in the past two years, according to a new report from the European Centre for Disease Prevention and Control (ECDC), which covers the European Union, Iceland, Liechtenstein, and Norway.

A new strain of influenza A, known as H3N2 subclade K, is driving the spread of the virus, the agency said. That’s the same variant cropping up in places like the United Kingdom, where there have also been signs of an earlier, tougher flu season.

While it’s not yet clear how severe the upcoming flu season will be in mainland Europe, the ECDC warned that it could be worse than previous years if there are more infections than usual and vaccine uptake remains low.

“We are seeing influenza detections increasing much earlier than usual this year, and that means time is critical,” Edoardo Colzani, a respiratory virus expert at the ECDC, said in a statement.

“Getting vaccinated now is one of the most effective ways to protect yourself and those around you from serious illness this winter,” he added.

The ECDC recommended vaccination “without delay” for older adults, pregnant women, people with chronic health issues or compromised immune systems, health workers, and those living in long-term care or other group settings.

Flu vaccines are updated every year to try to keep up with the virus, which is constantly evolving. An H3N2 strain was included in this year’s jab because it was one of the main viruses spreading in the Northern Hemisphere last season.

However, subclade K is different enough from that strain that it isn’t yet clear how well the vaccine will work against it.

Even so, health officials said vaccination is an important way to minimise illnesses, protect vulnerable groups, and reduce the burden on hospitals over the winter.

“If you are eligible for vaccination, please don’t wait,” Colzani said.

The ECDC also said that medical clinics and long-term care facilities should take additional steps to prevent and control infections, for example, by asking staff and visitors to wear face masks when the virus is spreading at higher levels.

More: https://www.msn.com/en-gb/health/other/flu-spreading-unusually-early-in-europe-amid-new-strain-health-officials-warn/ar-AA1QOFt7

Porous face masks have never been effective at stopping infections spread by aerosolised pathogens. Vitamin D, etc., is much better at warding off infection and lessening duration and severity.25

It’s clear that some professionals, who should know better, never learned any lessons from the scamdemic about NPIs or how the immune system works.

Hospitals roll out flu plan and facemasks ahead of tricky winter

Facemasks have been reintroduced in Lincolnshire hospitals as the trust braces for a difficult flu season. Cases are already high – particularly in children – and experts think the virus will be unusually bad this winter.

Patients are being asked to wear face masks in emergency departments, Urgent Treatment Centres and admission wards. Natalie Vaughan, the deputy director for infection prevention and control at United Lincolnshire Teaching Hospitals NHS Foundation Trust, said the virus would continue to be a problem.

“We look at places like Australia which have already had their winter, and their flu season was quite challenging. They had lots of cases and it came early,” she told the Local Democracy Reporting Service.

“That’s what we’re seeing happen here too now, in terms of people coming down with the flu and being hospitalised. We expect to see this increasing through the winter months, and are assessing it on a day-by-day basis.”

The flu virus mutates each year, and public health experts say this year’s strain is likely to be the most contagious for some time. Ms Vaughan’s message is to follow the key prevention strategies that became routine during the Covid pandemic – facemasks, hand hygiene and vaccination.

More: https://www.msn.com/en-gb/health/other/hospitals-roll-out-flu-plan-and-facemasks-ahead-of-tricky-winter/ar-AA1QPOnd

3.2 Wes is back, and boy, is he in trouble

Talk about being “out of his depth”.

First, from the 12th of November, he denied organising a coup against the PM:

And then three days later, he’s making the headlines again, for all the wrong reasons.

“The report also accuses the health secretary of pulling the NHS in contradictory directions, for example, by saying he wants to shift more care into the community but then proposing new “integrated health organisations”, which could end up allocating more money to hospitals.

The report adds that Streeting has provided very little detail on how his plans would be implemented, beyond relying on emerging technology such as voice-capture tools to take notes for GPs.”

https://www.theguardian.com/politics/2025/nov/15/wes-streeting-accused-of-chaotic-and-incoherent-approach-to-nhs-reform

I can’t see him being in this post for much longer, and I will miss his particular talent for muppetry.

4.0 Concluding remarks

Innocuous-looking tablets. What have we learned from over forty years of research?

Well, to start with, most of the regulators learned nothing ($$$), but the reality is grim, and you could even describe its promotion as “dark” or “sinister”.

I thought I had a fair understanding of APAP toxicity, but I was wide of the mark. As with statins, this isn’t just a fast-acting poison; it’s subtle and has dose-dependent toxic effects on the neurodevelopment of the foetus and newborns. Cancer patients also have their life expectancy cut short, perhaps without ever knowing the truth.

This is happening too frequently to be blamed on coincidence or “grandfather rights” for prescription-free approval. It also doesn’t get childhood vaccines off the hook, such as the completely pointless, but harmful Hep-B shots for babies:

To summarise the research findings:

• Acetaminophen was thought to be a safer alternative to acetanilide and phenacetin.

• Liver toxicity of APAP was first suspected in 1966.

• APAP toxicity is the second most common cause of liver transplantation worldwide and the most common cause of liver failure in the United States.

• It’s responsible for 56,000 emergency department visits, 2600 hospitalisations, and 500 deaths annually in the United States.

• Around 50% of these poisonings are unintentional.

• Despite limits on pack size, susceptible individuals can still take a toxic dose at the recommended clinical rate per day.

• The toxic metabolite NAPQI leads to glutathione depletion, binds with mitochondrial proteins, disrupts the ETC, leading to the generation of ROS, with hepatic necrosis as a consequence.

• Activated charcoal and NAC are first-line treatments.

• APAP freely crosses the placental barrier.

• NSAIDs such as acetaminophen have teratogenic effects through ROS generation, and lead to multifold increases in the risk of stillbirth, ASD, ADHD, bone and digit malformation, asthma, cryptorchidism and male infertility.

• There are many dietary sources that support glutathione status or contain the tripeptide itself.

• CD4, NK and NKT cells all play a part in mediating APAP-induced liver damage.

• APAP skews our immune system towards a proinflammatory Th1 state. This doesn’t help to protect us from cancer, though, because it also promotes PD1 expression and Treg infiltration of tumours.

• APAP causes dysfunction of cell-cell tight junction (TJ) adhesions. This is linked to the pathogenesis of various diseases, including inflammatory bowel disease (IBD), celiac disease, allergic conditions like asthma, atopic dermatitis, periodontal disease, cancer progression, and liver diseases such as cirrhosis and NAFLD.

• Immune response to vaccines is suppressed by APAP, and we also become more prone to many types of infection, which take longer to clear.

• For cancer patients, the prognosis is much poorer due to the blunted efficacy of checkpoint inhibitors and chemotherapy. Corticosteroids, in combination with APAP, shorten your life expectancy further still.

• Male fertility is reduced, too, as testosterone production is diminished.

• Excretion of acetaminophen in urine is a hazard to the aquatic environment.

• APAP leads to neurodevelopment disorders in children, partly due to endocannabinoid (eCB) dysfunction.

• Biomarkers for multi-organ developmental toxicity show that organ damage occurs even at therapeutic doses in healthy people. This presents a particular risk to susceptible individuals.

• Suppression of the Wnt signalling system by APAP is linked to loss of collagen and osteoporosis. This also affects the developing foetus.

• Many researchers and the FDA advise that pregnant women should avoid taking acetaminophen, especially for routine low-grade fevers. This is mainly to protect their offspring’s neurodevelopment.

Thank you for reading.

And if all this gave you a headache, I would consider taking something other than acetaminophen—magnesium would be a good alternative.

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5.0 Disclaimer

This site is strictly an information website reviewing research into potential therapeutic agents. It does not advertise anything, or provide medical advice, diagnosis, or treatment. This site does not promote any of these as potential treatments or offer any claims for efficacy. Its content is aimed at researchers, registered medical practitioners, nurses, or pharmacists. This content is not a substitute for professional medical advice, diagnosis, or treatment. Always seek the advice of your physician or other qualified health provider with any questions you may have regarding a medical condition. Never disregard professional medical advice or delay in seeking it because of something you have read on this website. Always consult a qualified health provider before introducing or stopping any medications as any possible drug interactions or effects will need to be considered.

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6.0 References

short story - novelette - novella - novel - PhD thesis - Trump’s tariff list - War and Peace - U.S. Tax Code

Any extracts used in the following article are for non-commercial research and educational purposes only and may be subject to copyright from their respective owners.

Contents

1.0 Background

2.0 Introduction

• 2.1 Chestnut honey

  • Honey, polyphenols and neurological disorders

  • Propolis

• 2.2 Kynurenic acid (KYNA)

  • Kynerenic acid and the kynurenine pathway

  • From chestnut honey to motor neurone disease

• 2.3 Other studies

  • Honey and osteoporosis

  • Kynurenic acid and cancer

  • Antimicrobial properties and wound healing

  • Kynurenic acid, the gut-brain axis and appetite

  • Kynurenic acid and neurological conditions

  • Kynurenic acid, chestnut honey, IBS and gastritis

3.0 Parting shots

• 3.1 Rotten to the core

• 3.2 Cognitive dissonance

• 3.3 The Future of Healthcare

• 3.4 Honey isn’t healthy

4.0 Concluding remarks

• 4.1 Dosing and other advice

5.0 Disclaimer

6.0 References

1.0 Background

In June this year, I was lucky enough to visit a honey farm on the French island of Corsica. Located near Ajaccio on maquis-covered slopes, “Le Jardin des Abeilles”, or Garden of Bees, has over 400 hives and produces 6 types of AOP-certified honey. Their Corsican bees don’t change throughout the year, but their nectar source changes with the season.

Spring honey is collected from March to May. This has a gorgeous floral taste, with low acidity. But what prompted me to write this Substack was the second honey sample I taste-tested. Corsican chestnut trees (also known as Spanish chestnut, or European chestnut, Castanea sativa) flower from early June to the end of July, and chestnut honey is collected from July to September.

Of all the honeys I’ve sampled as part of making mead, Chestnut honey was the most unusual, so far. Valued for cooking, it was the least favourite of the six being sampled by the tasters on account of its tannic nature, with bitter overtones. I wouldn’t wish to risk fermenting a whole batch of it!

The “Visit Corsica” site describes the natural progression of their honeys:

The rich wild natural vegetation and the ancestral knowledge of our beekeepers combine to create honey that is very particular. This product is made up of six seasonal varieties:

- Spring honey has a floral taste and lacks acidity. It is collected from March to May and has floral notes thanks to the asphodel and the clementine trees…

- The « Maquis de printemps » honey is essentially made from white heather, collected throughout the springtime across the vast expanses of the maquis scrubland that stretches from the sea to the mountains. It is golden, with a delicate caramel or cocoa flavour, with an aroma of liquorice or coconut.

- Maquis honeydew (cistus, oak, mastic), collected from May to September on the coastal zones and wooded areas close to the maquis, has a malty taste that lingers on the tongue, scented with liquorice, caramel and forest fruits.

- The « Maquis d’été » is gathered from the month of August and provides a fruity and aromatic honey, mixing helicrysum, anthyllis, Corsican thyme (erba barona), broom, marine germander and the aromatic plants of the mountains.

- Honey from the chestnut groves is collected from July onwards and is strong and tannic, with a slight bitterness. Its chestnut flavour has hints of bramble, anthyllis, clematis, broom and marjoram.

- The « Maquis d’automne » is bitter and strong in the mouth, collected from October to February and composed primarily of arbutus.

More: https://www.visit-corsica.com/en/content/view/full/38782

Mouthwatering?

For flavour, their “Maquis de printemps” honey was exquisite and is highly sought after. It was the one honey in their shop that was only available in small jars. Having said that, I consider their chestnut honey to be the real star of the show, at least as far as therapeutic potential is concerned.

2.0 Introduction

Manuka honey is derived from the Manuka tree (Leptospermum scoparium), which is grown exclusively in New Zealand and some parts of Australia. It is valued for its antibacterial and wound-healing properties, largely due to its high phenolic and methylgloxal (MGO) content1. Together, these form an antioxidant complex with anti-inflammatory and neuroprotective properties.2

So how does chestnut honey compare to this super-food, at a fraction of the cost? Read on…

2.1 Chestnut honey

Chestnut honey is derived from the nectar and honeydew (a sugary secretion from aphids that feed on the tree) of the chestnut tree. In the Mediterranean area, as with Corsica, forest groves of the Sweet Chestnut (Castanea sativa) are usually located between 1300’ and 3280’ (400 and 1000 m).

Centres of honey production around the Med include Southern France, Spain, Portugal, Italy, mainland Greece (Mount Athos, the mountains of Taygetus and Parnon, the highlands of Epirus) and Turkey. Globally, wherever you find native chestnut trees, you are likely to find their version of the honey, too.

The taste varies according to location and if the bees have also visited neighbouring pine or oak trees for honeydew, whilst on their travels. A bee may fly as far as five to seven miles in search of nectar, but a mile is more typical, and they will happily exploit any “target of opportunity” that presents itself.3

Colour can range from golden brown to almost black. I’ve consumed a 500g jar of Greek chestnut honey and found it to be much less bitter than the Corsican version, whilst being of similar appearance.

Honey, polyphenols and neurological disorders

(Some abstracts are lightly re-formatted with paragraph breaks for legibility on mobile devices)

Potential Therapeutic Benefits of Honey in Neurological Disorders: The Role of Polyphenols (2022)

… Honey is the primary product of apiculture with a history of use corresponding to the history of mankind. The nutritional and therapeutic benefits of honey have been indicated in every culture and religion of the world including Greek, Roman, Christianity, and Islam [1,2].

Physicians of ancient times have extensively discussed the medicinal qualities of honey. Ancient Egyptian physicians employed honey in their medication 5000 years ago and the prehistoric Greeks believed honey beneficial for vigor and longevity. The first written reference to the beneficial effects of honey, a Sumerian tablet writing, dating back to 2100–2000 B.C., indicates honey as a potential drug to be used against various complaints.

… 2. Composition of Honey

The beneficial effects of honey are attributed to various biological bioactive components. The presence of these active compounds justifies the substantial biological benefits of honey. The percentage of all these components varies among different types of honey. In general, honey consists of more than 200 substances. It is mainly a carbohydrate product, and sugars constitute more than 90% of solids. Main sugars found in honey include glucose, sucrose, maltose, fructose, melezitose, isomaltose, maltulose, turanose, nigerose, melibiose, panose, and maltotriose.

Water is the second most important component of honey. Conjointly with carbohydrates and water, honey also contains enzymes, vitamins, minerals, flavonoids, and polyphenols [7,8,9]. Riboflavin (Vit. B2), Niacin (Vit. B3), Pantothenic acid (Vit. B5), Pyridoxine (Vit. B6), Folate (Vit. B9), and vitamin C are the major vitamins found in honey.

Among minerals, potassium is the major one while calcium, magnesium, sodium, sulfur, and phosphorus are also found in a significant amount. The main enzymes found in honey include invertase (saccharase), diastase (amylase), and glucose oxidase. Non-enzymatic proteins, including glycoprotein, MRJP1, and apalbumin-1, are also found in honey, but in very minute quantities [9].

Polyphenols are a major class of naturally occurring organic compounds that are defined by multiples of phenol units. Polyphenols consist of flavonoids and nonflavonoids that are separated into subclasses based on the number of phenol units, substituent groups, and/or the kind of linkage between phenol units in their molecular structure. A 15-carbon (C6–C3–C6) backbone with two phenyl units (A and B), along with a heterocyclic unit (C), is the characteristic of flavonoids.

Non-flavonoid polyphenolic compounds include phenolic acids, coumarins, lignans, hydrolyzable tannins, lignins, and condensed tannins. Honey contains a wide range of both flavonoids and phenolic acids [10].

Among flavonoids, quercetin, myricetin, kaempferol, luteolin, rutin, naringenin, naringin, chrysin, rhamnetin, isorhamnetin, apigenin, pinocembrin, pinobanksin, galangin, tricetin, catechin, and hesperidin are the major compounds found in various varieties of honey.

The most common phenolic acids, occurring in almost every honey type, include caffeic acid, gallic acid, coumaric acid, syringic acid, cinnamic acid, ferulic acid, chlorogenic acid, ellagic acid, benzoic acid, vanillic acid, phenylacetic acid, and homogentisic acid.

… The leading underlying cause of nerve death is oxidative stress caused by an accumulation of free radicals and depletion of antioxidants [86]. Increasing the levels of antioxidants can be beneficial against these neurodegenerative diseases [24].

Honey contains antioxidants in the form of polyphenols and flavonoids. All kinds of honey contain these bioactive compounds but in varying concentrations. Various colorimetric assays reveal that the total phenolic content in various types of honey varies from 86 mg/kg to 1141 mg/kg [87], whereas the range for the flavonoid content is from 36 mg/kg to 150 mg/kg of honey [88].

Both flavonoids and polyphenols protect neurons against oxidative damage, improve neuronal function and enhance regeneration, protect neurons from neurotoxicity, and modulate neuronal signaling pathways [24].

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC9143627/

An analysis of Turkish chestnut honey from 41 locations quantified the presence of 16 phenolic compounds. Their relative proportions varied with geographical location and nectar sources.

Chemometric classification of chestnut honeys from different regions in Turkey based on their phenolic compositions and biological activities (2023)

Highlights

• Phenolic composition and bioactivities of Turkish chestnut honeys were investigated.

• The presence of 16 phenolic compounds in chestnut honeys was detected by HPLC-DAD.

• Chemometric analysis (PCA and HCA) was used to classification the chestnut honeys.

• The phenolic composition depends on the botanical origin of chestnut honeys.

… 3.2. Phenolic compounds

The phenolic compounds and organic acids composition of chestnut honey samples were depicted in Table 2. Twenty-six phenolic compound and organic acid standards were analyzed, but sixteen standard compounds (levulinic acid, gallic acid, protocatechuic acids, p-hydroxy phenylacetic acid, (4-hydroxyphenyl) ethanol, p-hydroxybenzoic acid, 6,7-dihydroxy coumarin, vanillic acid, p-coumaric acid, ferulic acid, rutin, 2-hydroxy cinnamic acid, rosmarinic acid, quercetin, trans-cinnamic acid and chrysin) were detected in chestnut honey samples.

Levulinic, gallic, protocatechuic, vanilic, trans-cinnamic acids and (4-hydroxyphenyl) ethanol were identified in all studied chestnut honey samples. Gallic acid was detected as the major compound in all chestnut honeys studied, except for BO, BI, BK1 BK2, KS, KC1, KD1, KD2, KC2, KI1 and SG coded samples.

The gallic acid content of chestnut honey samples ranged from 4.08 to 183.74 μg/g. p-Hydroxy phenylacetic acid was found to be the main compound in samples BO (120.38 μg/g), BI (115.60 μg/g), BK1 (111.20 μg/g) and BK2 (86.65 μg/g) while rutin was the major compound in KS (80.55 μg/g), KC1 (72.43 μg/g), KD2 (65.64 μg/g), KD1 (52.20 μg/g), KC2 (49.00 μg/g), KI1 (34.31 μg/g) and SG (29.93 μg/g) samples. 2-Hydroxy cinnamic acid, rosmarinic acid and quercetin were determined only in Bursa of BO, BI, BK1, BK2, BOS, and BIN chestnut honey samples.

While p-coumaric acid (0.01 μg/g) was not found at all in samples from Bursa, it was found in small amounts in all other chestnut honey samples. Protocatechuic acid (0.01 μg/g) was found in small quantities in all studied chestnut honey samples except AA (0.69 μg/g), IS (0.96 μg/g), IB (1.03 μg/g), SSAP (1.48 μg/g), ZA (0.21 μg/g) and ZC (0.38 μg/g) coded samples (Table 2).

… 4. Conclusions

Forty-one chestnut honey samples from 8 different regions (Black Sea and Marmara) in Turkey were classified according to the amounts of phenolic compounds, antioxidant, antimicrobial, anti-inflammatory, anti-urease, anticholinesterase and anti-tyrosinase activities using chemometric approach.

In this study it has been revealed that phenolic compounds can be used as a distinguishing factor according to the regions where chestnut honey is produced, and the phenolic content of honey differs qualitatively and quantitatively depending on the botanical origin and nectar sources of honey.

It is concluded that levulinic, gallic, vanilic, trans-cinnamic acids and (4-hydroxyphenyl) ethanol could be used as marker compounds in Turkish chestnut honeys. Studied chestnut honeys possesses significant antioxidant, antimicrobial, anti-quorum sensing, anti-inflammatory, anticholinesterase, anti-urease and anti-tyrosinase activities that can be used as functional food in complementary and supportive theraphy. In this way, chestnut honeys may be useful in various applications such as food, cosmetics and medicine.

More: https://www.sciencedirect.com/science/article/pii/S0308814623003448?via%3Dihub

DNA repair. Very useful for delaying the effects of ageing and for cancer prevention.

(Paywalled):

Phenolic profiles, antioxidant, antimicrobial, and DNA damage inhibitory activities of chestnut honeys from Black Sea Region of Turkey (2018)

Abstract

Honey is the product of beekeeping that has great market potential thanks to valuable nutritional and medicinal qualities. Black Sea Region coast of Turkey is suitable for production of chestnut production so chestnut-based honeys are highly produced in this region.

To prove the beneficial therapeutic properties, 49 chestnut (Castania sativa Mill.) honey samples from the cities of Black Sea Region were investigated in terms of colors, total phenolic and flavonoid contents, antioxidant activities, antimicrobial potentials, phenolics, and effects on hydroxyl radical-induced DNA breaks in the non-site-specific system.

The amount of flavonoid was found to be limited in chestnut honey samples compared to the phenolic acids. A wide variation was observed in the amounts of total antioxidant activities of honey samples. It was displayed that most of the tested honey samples had the ability to repair the DNA breaks created by hydroxyl radicals.

More: https://onlinelibrary.wiley.com/doi/abs/10.1111/jfbc.12502

This paper discusses the properties of higher-quality chestnut honeys.

Sinop is a peninsula in northern Turkiye, on the Black Sea Region coast. The chestnut-covered hills are around 1500 - 2000’ / 457 - 610m high.

(Paywalled):

Determination of the superior quality properties of randomly selected chestnut honey samples from the Sinop region (2023)

Abstract

In this study, a total of 43 chestnut honey samples were randomly selected and collected from the Erfelek, Ayancik, and Turkeli districts of Sinop. The pollen content of all honey samples was determined, revealing that chestnut pollen accounted for over 90% in most cases. The chestnut honey from the Sinop region is considered of high quality compared to other chestnut honeys, primarily due to its low HMF (Hydroxymethylfurfural) levels, low moisture content, high proline content, and significant pollen content.

More: https://acikerisim.sinop.edu.tr/items/5ffb25bc-7493-4127-ad30-3ed1f9d70c73

A polyphenol-rich diet helps to prevent insulin resistance and diabetes. This, in turn, helps to slow the rate of age-associated cognitive decline.

Glycemic control contributes to the neuroprotective effects of Mediterranean and green-Mediterranean diets on brain age: the DIRECT PLUS brain-magnetic resonance imaging randomized controlled trial (2024)

Abstract

Background

We recently reported that Mediterranean (MED) and green-MED diets significantly attenuated age-related brain atrophy by ∼50% within 18 mo.

Objective

The objective of this study was to explore the contribution of specific diet-induced parameters to brain-volume deviation from chronologic age.

Methods

A post hoc analysis of the 18-mo DIRECT PLUS trial, where participants were randomly assigned to the following groups: 1) healthy dietary guidelines, 2) MED diet, or 3) green-MED diet, high in polyphenols, and low in red meat. Both MED groups consumed 28 g walnuts/d (+440 mg/d polyphenols). The green-MED group further consumed green tea (3–4 cups/d) and Mankai green shake (Wolffia globosa aquatic plant) (+800 mg/d polyphenols). We collected blood samples through the intervention and followed brain structure volumes by magnetic resonance imaging (MRI). We used hippocampal occupancy (HOC) score (hippocampal and inferior lateral-ventricle volumes ratio) as a neurodegeneration marker and brain-age proxy. We applied multivariate linear regression models.

Results

Of 284 participants [88% male; age = 51.1 y; body mass index = 31.2 kg/m²; hemoglobin A1c (HbA1c) = 5.48%; APOE-ε4 genotype = 15.7%], 224 completed the trial with eligible whole-brain MRIs. Individuals with higher HOC deviations (i.e., younger brain age) presented lower body weight [r = –0.204; 95% confidence interval (CI): –0.298, –0.101], waist circumference (r = –0.207; 95% CI: –0.310, –0.103), diastolic (r = –0.186; 95% CI: –0.304, –0.072), systolic blood pressure (r = –0.189; 95% CI: –0.308, –0.061), insulin (r = –0.099; 95% CI: –0.194, –0.004), and HbA1c (r = –0.164; 95% CI: –0.337, –0.006) levels.

HbA1c: Glycated haemoglobin, a measure of blood glucose and diabetes.

“Normal”= below 42-47 mmol/mol.

HOMA-IR: Homeostatic Model Assessment for Insulin Resistance. Lower is better.

c-reactive protein: A blood marker for systemic inflammation. Produced by the liver.

After 18 mo, greater changes in HOC deviations (i.e., brain-age decline attenuation) were independently associated with improved HbA1c (β = –0.254; 95% CI: –0.392, –0.117), HOMA-IR (β = –0.200; 95% CI: –0.346, –0.055), fasting glucose (β = –0.155; 95% CI: –0.293, –0.016), and c-reactive protein (β = –0.153; 95% CI: –0.296, –0.010).

Improvement in diabetes status was associated with greater HOC deviation changes than either no change in diabetes status (0.010; 95% CI: 0.002, 0.019) or with an unfavorable change (0.012; 95% CI: 0.002, 0.023).

A decline in HbA1c was further associated with greater deviation changes in the thalamus, caudate nucleus, and cerebellum (P < 0.05). Greater consumption of Mankai and green tea (green-MED diet components) were associated with greater HOC deviation changes beyond weight loss.

Conclusions

Glycemic control contributes to the neuroprotective effects of the MED and green-MED diets on brain age. Polyphenols-rich diet components as Mankai and green tea may contribute to a more youthful brain age.

More: https://www.sciencedirect.com/science/article/pii/S0002916524007457

Many Turkish people take honey daily as part of their culture. This is often, quite rightly, for its health benefits—typically one to two tablespoons mixed with butter.4

Glycemic index values of monofloral Turkish honeys and the effect of their consumption on glucose metabolism (2016)

Abstract

Background/aim: Clinical trials have shown that low glycemic index (GI) nutrition reduces mean blood glucose concentrations and insulin secretions. The aim of the present study was to determine the GI values of various monofloral (citrus, milk-vetch, chestnut, thyme, lime, pine) honeys of Turkey, and the effect of their consumption on glucose metabolism.

Materials and methods: Processing data from 20 healthy volunteers, GI values were determined from the glycemia values by using the incremental area method. Serum insulin and C-peptide levels were also measured before and 120 min after the test.

Results: The GI values of citrus, thyme, lime, chestnut, pine, and milk-vetch honeys were found to be 44.9, 52.6, 55.3, 55.5, 58.8, and 69, respectively. Serum insulin and C-peptide values after honey consumption were relatively lower than those after reference food (glucose) consumption. By the end of the 120 min, serum insulin levels were significantly higher, while a significant decrease was observed after the consumption of chestnut honey (P < 0.05).

Conclusion: Citrus and thyme honeys were determined to have low GI, while serum insulin levels were significantly lower after the consumption of chestnut honey. Long-term research is needed to compare the effects of honey consumption on healthy and diabetic individuals.

Keywords: Honey; glucose metabolism; glycemic index; nutrition.

... Additionally, it is interesting that serum insulin levels were determined to be significantly low 2 h after chestnut honey consumption, while a significant increase was observed after reference food consumption.

More: https://journals.tubitak.gov.tr/medical/vol46/iss2/37/

This study, conducted in mice, demonstrated that supplementation with chestnut honey may help prevent neuronal damage caused by a high-fat diet.

Preventive Impact of Long-Term Ingestion of Chestnut Honey on Glucose Disorders and Neurodegeneration in Obese Mice (2022)

Abstract

The purpose of the present study was to evaluate the impact of long-term honey ingestion on metabolic disorders and neurodegeneration in mice fed a high-fat diet (HFD). Three groups of mice were fed with a standard diet (STD), HFD or HFD supplemented with honey (HFD-H) for 16 weeks. Biochemical, histological, Western blotting, RT-PCR and Profiler PCR array were performed to assess metabolic parameters, peripheral and central insulin resistance and neurodegeneration.

Daily honey intake prevented the HFD-induced glucose dysmetabolism. In fact, it reduced plasma fasting glucose, insulin and leptin concentrations and increased adiponectin levels. It improved glucose tolerance, insulin sensitivity and HOMA index without affecting plasma lipid concentration.

HFD mice showed a significantly higher number of apoptotic nuclei in the superficial and deep cerebral cortex, upregulation of Fas-L, Bim and P27 (neuronal pro-apoptotic markers) and downregulation of Bcl-2 and BDNF (anti-apoptotic factors) in comparison with STD- and HFD-H mice, providing evidence for honey neuroprotective effects.

PCR-array analysis showed that long-term honey intake increased the expression of genes involved in insulin sensitivity and decreased genes involved in neuroinflammation or lipogenesis, suggesting improvement of central insulin resistance.

The expressions of p-AKT and p-GSK3 in HFD-H mice, which were decreased and increased, respectively, in HFD mouse brain, index of central insulin resistance, were similar to STD animals supporting the ability of regular honey intake to protect brain neurons from insulin resistance. In conclusion, the present results provide evidence for the beneficial preventative impact of regular honey ingestion on neuronal damage caused by HFD.

Keywords: HFD mice; honey; insulin resistance; neurodegeneration; obesity.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC8879402/

The following year, the same authors conducted follow-up research using limonene (from Citrus) in combination with chestnut honey and found synergistic effects.

Long-Term Ingestion of Sicilian Black Bee Chestnut Honey and/or D-Limonene Counteracts Brain Damage Induced by High Fat-Diet in Obese Mice (2023)

… We analyzed the brain neurodegeneration, inflammation, oxidative stress, and gene expression of Alzheimer’s disease (AD) markers. The HFD animals showed higher neuronal apoptosis, upregulation of pro-apoptotic genes Fas-L, Bim P27 and downregulation of anti-apoptotic factors BDNF and BCL2; increased gene expression of the pro-inflammatory IL-1β, IL-6 and TNF-α and elevated oxidative stress markers COX-2, iNOS, ROS and nitrite.

The honey and D-limonene intake counteracted these alterations; however, they did so in a stronger manner when in combination. Genes involved in amyloid plaque processing (APP and TAU), synaptic function (Ache) and AD-related hyperphosphorylation were higher in HFD brains, and significantly downregulated in HFD-H, HFD-L and HFD-H + L. These results suggest that honey and limonene ingestion counteract obesity-related neurodegeneration and that joint consumption is more efficacious than a single administration.

… Recently, D-limonene (1-methyl-4-(1-methylethenyl) cyclohexane), a monocyclic monoterpene that is the major constituent of citrus essential oils, has also received notable scientific interest due to its ability to mitigate inflammation and oxidative stress and reduce apoptotic cell death [24].

In fact, it possesses antidiabetic, antioxidant, anti-inflammatory, antinociceptive and anticancer properties [25]. In animal models, D-limonene has been reported to alleviate obesity-related metabolic disorders [26,27].

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC9966634/

The ethyl acetate content of chestnut honey also helps to prevent neurotoxicity.

Ethyl Acetate Fraction of Chestnut Honey Attenuates Scopolamine-Induced Cognitive Impairment in Mice and Glutamate-Induced Neurotoxicity in HT22 Cells (2024)

Chestnut honey has various benefits, such as antioxidative, anti-inflammatory, immunomodulatory, antibacterial, and antiviral effects. However, the effects of chestnut honey or the ethyl acetate fraction of chestnut honey (EACH) on neurodegenerative diseases and their related cognitive impairment and neurotoxicity have not yet been established.

Therefore, in this study, we investigated the mitigating effect of the EACH on scopolamine (SCO)-injected cognitive decline in mice and glutamate-exposed neurotoxicity in HT22 cells. EACH administration significantly reversed SCO-induced cognitive decline in mice, as demonstrated through the Morris water maze and passive avoidance tests. The EACH treatment showed a significant alleviation effect by recovering more than 80% of the cell viability decrease induced by glutamate exposure in the HT22 neuronal cell model.

Furthermore, the EACH significantly reduced reactive oxygen species accumulation, lactate dehydrogenase release, mitochondrial depolarization, and neuronal apoptosis.

The EACH regulated the level of apoptosis-related proteins, induced the nuclear translocation of nuclear factor-E2-related factor 2 (Nrf-2) and the expression of related antioxidant proteins, and induced the phosphorylation of tropomyosin-related kinase receptor B (TrkB)/cAMP-calcium response element-binding protein (CREB) and the expression of brain-derived neurotrophic factor.

These data indicate that the EACH can prevent neurons from oxidative damage and improve cognitive dysfunction by activating Nrf-2 and TrkB/CREB signaling pathways. Therefore, the EACH demonstrates potential therapeutic value in mitigating oxidative stress-induced neurotoxicity, cognitive decline, and related neurodegenerative diseases.

… the group that was administered with EACH spent more time in the NE quadrant, and the number of times they crossed the platform location was also significantly higher than that in the SCO group (Figure 2A–C). However, the 100 mg/kg EACH group showed statistically significant efficacy in both parameters, but the 300 mg/kg EACH group did not, so no dose dependency was observed.

To evaluate the working memory of the mice in each group, the PA test was performed 2 days after the probe test, and the test trial was conducted 1 day after the training trial. The EACH group showed a statistically significant increase in the average step-through latency compared with the SCO group at both the 100 and 300 mg/kg doses (Figure 2D).

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC11591166/

This study demonstrated how honeys may be beneficial for treating bacterial infections, inflammation and sepsis.

Anti-Inflammatory and Antibacterial Effects and Mode of Action of Greek Arbutus, Chestnut, and Fir Honey in Mouse Models of Inflammation and Sepsis (2022)

… Results: The administration of all Greek honey samples to LPS-stimulated mice revealed a potent anti-inflammatory activity by suppressing the TNFα serum levels and the expression of TNFα and iNOS in the liver at levels comparable to those of the manuka honey, but they had no effect on IL-6 or IL-1β.

It was shown that the LPS-induced suppression of CYP1A1 in the liver was reversed by Epirus and Crete fir honey, while, correspondingly, the suppression of CYP2B10 in the liver was reversed by Evros chestnut and Epirus fir honey. The effect of the same honey samples in polymicrobial peritonitis in mice was also evaluated.

Even though no effect was observed on the disease severity or peritoneal bacterial load, the bacterial load in the liver was reduced in mice treated with Evros chestnut, Epiros fir, and Crete fir, while the bacterial load in the lungs was reduced in Epirus arbutus, Crete fir, and manuka honey-treated mice.

Conclusion: Our findings suggest that these specific Greek honey samples possess distinct anti-inflammatory and antibacterial properties, as evidenced by the reduced production of pro-inflammatory mediators and the impaired translocation of bacteria to tissues in septic mice. Their mode of action was comparable or more potent to those of manuka honey.

… The administration of honey reduced the bacterial load in the tissues of septic mice. This could be an indication that honey has an important role in preventing translocation of microorganisms. In accordance with this finding, honey was previously found to reduce bacterial translocation rates from the intestinal lumen to mesenteric lymph nodes or other extraintestinal tissues in an experimental obstructive jaundice model [52,53].

… 5. Conclusions

In conclusion, our results showed that treatment with Greek arbutus, chestnut, and fir honeys resulted in the suppression of inflammatory responses in mice with acute endotoxemia by reducing the production of TNF-α and iNOS. In addition, the administration of Greek honeys decreased the bacterial translocation to tissues during polymicrobial peritonitis.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC9784341/

Propolis

(Not an endorsement):

Propolis is a natural substance that bees make by gathering resin from cone-producing evergreen trees or from the buds of trees such as the Poplar.
Propolis has been used for thousands of years as a natural antiseptic and antibiotic and its medical benefits are well documented.

https://baxbees.co.uk/shop/propolis/raw-propolis/

Monoamine oxidase (MAO) is another component of chestnut honey with neuroprotective properties.

Total monoamine oxidase (MAO) inhibition by chestnut honey, pollen and propolis (2013)

Abstract

Monoamine oxidase (MAO) inhibitors are generally used in the treatment of depressive disorders and some neurodegenerative illnesses, such as Parkinson’s disease and Alzheimer’s disease. The aim of this preliminary study was to investigate the MAO [MAO (E.C.1.4.3.4)] inhibiting effect of various apitherapeutic products, such as chestnut honey, pollen and propolis.

Extracts’ MAO inhibition was measured using peroxidase-linked spectrophotometric assay in enzyme isolated from rat liver microsomes, and the values are expressed as the inhibition concentration (IC₅₀) causing 50% inhibition of MAO.

The antioxidant activity of the bee products was also determined in terms of total phenolic content (TPC) and ferric reducing/antioxidant power in aquatic extracts. All samples exhibited substantial inhibition of MAO, propolis having the highest. Inhibition was related to samples’ TPCs and antioxidant capacities. These results show that bee products possess a sedative effect and may be effective in protecting humans against depression and similar diseases.

More: https://www.tandfonline.com/doi/10.3109/14756366.2013.843171

Caffeic acid, a component of chestnut honey-based propolis, may also be used to treat Alzheimer’s disease.

(Paywalled):

Naturally occurring caffeic acid phenethyl ester from chestnut honey-based propolis and virtual screening towards DYRK1A (2023)

Abstract

Neurodegenerative diseases (NDDs) are disorders with dysfunction and ongoing loss of neurons, glial cells and the neural networks in the brain and spinal cord. DYRK1A protein was reported to modulate to the cytoskeletal fraction in human and mouse brain, and the remaining protein is located in the cytosolic and nuclear fractions. Caffeic acid phenethyl ester (CAPE) is a natural derivative of caffeic acid and found in propolis, a bee product.

In this study, we focused on isolation and characterisation of CAPE from chestnut honey-based propolis by HPLC-MS/MS technique and virtual screening of CAPE towards DYRK1A by molecular docking methods. Results revealed that CAPE might be a beneficial option to treat Alzheimer disease (AD) by suppressing DYRK1A protein.

Keywords: Alzheimer disease; CAPE; DYRK1A; neurodegenerative diseases; propolis.

More: https://www.tandfonline.com/doi/10.1080/14786419.2024.2309660

2.2 Kynurenic acid (KYNA)

Broadly speaking, KYNA is to chestnut honey what MGO is to Manuka.

Kynerenic acid and the kynurenine pathway

KYNA is a metabolite involved in the kynurenine pathway (KP). I discussed the link between disruption to the KP and Long COVID/Long COVID Vax syndrome back in 2022.

A recap from “Spike protein (inc vax) induced immunodeficiency & carcinogenesis megathread #29: The tryptophan catabolite or kynurenine pathway and long COVID”:

The kynurenine pathway is a metabolic pathway leading to the production of nicotinamide adenine dinucleotide (NAD+). Metabolites involved in the kynurenine pathway include tryptophan, kynurenine, kynurenic acid, xanthurenic acid, quinolinic acid, and 3-hydroxykynurenine. The kynurenine pathway is responsible for total catabolization of tryptophan about 95%. Disruption in the pathway is associated with certain genetic and psychiatric disorders.

Kynurenine pathway dysfunction

Disorders affecting the kynurenine pathway may be primary (of genetic origin) or secondary (due to inflammatory conditions). Peripheral inflammation can lead to a build up of kynurenine in the brain, and this is associated with major depressive disorder, bipolar disorder, and schizophrenia. Dysfunction of the pathway not only cause increasing metabolites such as quinolinic acid or kynurenic acid but also affects serotonin and melatonin synthesis. Kynurenine clearance in exercised muscle cells can suppress the build up in the brain.

Dysfunctional states of distinct steps of the kynurenine pathway (such as kynurenine, kynurenic acid, quinolinic acid, anthranilic acid, 3-hydroxykynurenine) have been described for a number of disorders, for example:

• HIV dementia

• Tourette syndrome

• Tic disorders

• Psychiatric disorders (such as schizophrenia, major depressive disorder, bipolar disorder, anxiety disorders)

• Multiple sclerosis

• Huntington's disease

• Encephalopathies

• Lipid metabolism

• Liver fat metabolism

• Systemic lupus erythematosus

• Glutaric aciduria

• Vitamin B6 deficiency

• Eosinophilia-myalgia syndrome

• Long COVID

Tryptophan (symbol Trp or W) is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic beta carbon substituent. It is essential in humans, meaning that the body cannot synthesize it and it must be obtained from the diet. Tryptophan is also a precursor to the neurotransmitter serotonin, the hormone melatonin, and vitamin B3. It is encoded by the codon UGG.

Like other amino acids, tryptophan is a zwitterion at physiological pH where the amino group is protonated (–NH+3; pKa = 9.39) and the carboxylic acid is deprotonated ( –COO−; pKa = 2.38).[5]

Humans and many animals cannot synthesize tryptophan: they need to obtain it through their diet, making it an essential amino acid

From chestnut honey to motor neurone disease

KYNA can be a marker for disease, but correlation doesn’t imply causation.

Key takeaways from “Kynurenic Acid: The Janus-Faced Role of an Immunomodulatory Tryptophan Metabolite and Its Link to Pathological Conditions” (2017) by Wirthgen et al.:5

• Tryptophan metabolites are known to participate in the regulation of many cells of the immune system and are involved in various immune-mediated diseases and disorders.

• Kynurenic acid (KYNA) is a product of one branch of the kynurenine pathway of tryptophan metabolism. The influence of KYNA on important neurophysiological and neuropathological processes has been comprehensively documented.

• In recent years, the link of KYNA to the immune system, inflammation, and cancer has become more apparent.

• Given this connection, the anti-inflammatory and immunosuppressive functions of KYNA are of particular interest. These characteristics might allow KYNA to act as a “double-edged sword.”

With most of the alternative therapeutics I’ve reviewed, this isn’t a concern because they either help to delay the onset of cancer in the first place, or they signal other anti-cancer pathways. However, it needs to be considered:

• The metabolite contributes to both the resolution of inflammation and the establishment of an immunosuppressive environment, which, for instance, allows for tumor immune escape.

• Within the central nervous system, it has been well documented that metabolites such as KYNA and QUIN modulate neurological functions. Thus, KYNA acts as an antagonist affecting all ionotropic glutamate receptors including NMDA, AMPA, and kainate receptors as well as the α7 nicotinic acetylcholine receptor (α7nAChR) assuming it as a neuroprotective metabolite (15–18).

Even now, the mode of action has not been confirmed experimentally:

• However, the inhibition of α7nAChR by KYNA is extensively debated because some later studies addressing this mechanism (3, 19–21) could not recapitulate the original results from Hilmas et al. (18).

• Dysregulation of KP, resulting in alterations of the balance between KYNA and QUIN, has been described in many neurological disorders (22).

Less inflammation is better with these conditions:

• However, alterations of KYNA are also described in several inflammatory-related states, such as sepsis or inflammatory bowel disease (IBD), and are discussed as a potential marker in cancer patients (23–25).

• It is generally accepted that KYNA mediates immunosuppressive effects (22), notably by targeting the G-protein-coupled receptor 35 (GPR35)- or aryl hydrocarbon receptor (AhR)-associated signaling pathways (2, 26, 27).

Pharmacokinetics and therapeutic mechanisms are challenging to demonstrate in the lab because of involvement with gut microbiota, in vivo. It’s not sufficient to use a simple in vitro model to test for therapeutic properties. We saw the same thing with many other therapeutics, such as with icariin, and especially with DMSO:

• Studies in rabbits described differences in KYNA metabolism depending on the type of administration (58). After oral administration of KYNA, the majority of the dose was detected in the form of quinaldic or 8-hydroxyquinaldic acid, indicating a dehydroxylation of the molecule.

• In contrast, after subcutaneous administration, 99% of KYNA was recovered unchanged, indicating that the dehydroxylation occurs in the gastrointestinal tract (58) most probably by the gut microbiota.

• This is supported by the finding that approximately 30% of ingested KYNA was excreted in urine as quinaldic acid in humans (59).

• The assumption that KYNA is metabolized by microorganisms is supported by the finding that extracts of Pseudomonas spp. and Aerococcus spp. were able to enzymatically partially degrade KYNA (60, 61).

There is a link between KYNA, the Gpr35 receptor, and osteoblast/osteoclast activity, too.

More KYNA is better:

LPS: lipopolysaccharide, the hyper-inflammatory endotoxin produced by many bacteria. LPS is linked to toxic shock and sepsis.

• The KYNA effect under inflammatory conditions appears to be more uniformly. Several in vitro studies, using various primary or immortalized leukocyte cell types, have revealed that KYNA can attenuate inflammation elucidated by different stimuli (e.g., LPS).

TNF: Tumor Necrosis Factor or TNF-alpha is a proinflammatory cytokine that, in the right context, may promote cancer growth:

• For instance, KYNA reduces TNF expression and secretion (26, 55, 65, 66) and diminishes high-mobility group box 1 (HMGB1) protein secretion in monocytes (66, 67).

α-defensin HNP1–3 is an antimicrobial neutrophil peptide. Elevated levels of HNP1-3 are associated with both type 1 and type 2 diabetes:6

Interleukin 4 (IL-4) may be both pro- or anti-inflammatory, depending on context.

• Likewise, KYNA has been shown to inhibit the secretion of α-defensin HNP1–3 in granulocyte cultures (66) and reduce interleukin 4 release in T-cell receptor stimulated invariant natural killer-like T cells (iNKT) (68).

Interleukin 23 or IL-23 is a pro-inflammatory cytokine, and is linked to the upregulation of pro-inflammatory IL-17. Almost all cytokines of this type are associated with the progression of many types of cancer and autoimmune disorders:

• Recently, Elizei et al. (69) demonstrated that KYNA reduced LPS-induced IL23 expression of dendritic cells and inhibited Th17 cell differentiation in vitro.

Chestnut honey should be beneficial for many patients with autoimmune conditions involving the IL23/IL17 axis, such as fibromyalgia syndrome (FMS),7 multiple sclerosis (MS) and irritable bowel syndrome (IBS)8:

• The downregulation of the IL23/IL17 axis is known to be beneficial for anti-inflammatory treatment of many immune-mediated diseases (70).

Disrupted KP and KYNA levels are associated with several progressive, severe neurological conditions:

• A link between inflammation and increased brain levels of KYNA was furthermore described in amyotrophic lateral sclerosis (ALS) patients. This study demonstrated a correlation between increased levels of KYNA in CSF with the severe clinical status of ALS (137).

AD: Alzheimer’s Disease.

• It is assumed that oxidative stress, glutamatergic excitotoxicity, or neuroinflammation play key roles in the pathophysiology of neurodegeneration, particularly in ALS or AD (148).

Note: KYNA isn’t contributing to these conditions; it’s attempting to mitigate their associated neurotoxicity:

• Therefore, the increased production of KYNA may act as compensatory response to neurotoxic effects. The fact that KYNA was decreased in the blood of patients with a severe clinical state of ALS (137) supports the suggestion that TRP or KYN from the periphery is used as precursors for increased brain synthesis of KYNA, knowing that KYNA is hardly able to cross the blood–brain barrier (149).

With schizophrenia, there is evidence for a genetic link to elevated levels of KYNA, according to this study:

• Increased levels of KYNA in saliva were also detected in schizophrenia patients with concurrent distress intolerance compared with distress-tolerant patients and healthy controls. This finding indicated an interference of stress with the activation of KP (129).

Some inherited diseases also have disrupted KYNA levels:

• In patients suffering from Huntington’s disease, KYNA was decreased in the CSF and several brain regions (135, 136, 150). It was suggested that this was due to a selective impairment in KYNA biosynthesis in specific brain areas of HD patients (150), resulting in an inadequate anti-inflammatory and neuroprotective response to inflammatory conditions.

• Increased concentrations of KYNA in the brain were described in patients with Down syndrome (133), which exhibit similar neuropathological features as patients with AD, such as neuritic amyloid-β plaques (151).

• This indicates that neuro-inflammatory processes may play a role in the Down syndrome phenotype. Increased brain levels of KYNA may reflect a compensatory response to neurotoxic effects due to congenital malfunctions.

• Furthermore, the finding that in Down syndrome patients the urinary excretion of KYN was lower concurrent with increased excretion of KYNA, and anthranilic acid, suggests a shift of KP to the neuroprotective and antioxidative branch (134).

The link between KYNA, the microbiome, colon cancer and IBS:

• High GPR35 expression in the gastrointestinal tract (26, 79) indicates that this receptor, and probably its ligand KYNA, could have a function in gut homeostasis (152).

• The potential significance of KYNA for gut health emerges from its association with various bowel diseases and colon cancer [Table 1 (30, 138, 139)], as well as the potential anti-inflammatory effects of KYNA treatment in dogs with experimental colon obstruction (71).

• Studies in rats and pigs have shown a high concentration of KYNA in the intestinal lumen (153, 154). The intestinal KYNA concentration increased from the proximal to the distal part of the gut, reaching ~16 μM in the distal ileum of the rat (153) and ~1.6 μM in the colon of the pig (154).

• The studies in rat suggest that relevant amounts of KYNA in the gut originated from the intestinal microflora, due to the relatively low concentrations in the wall of the ileum (~0.2–0.3 μM) and the food (~0.6 μM).

Yes, dietary intake of KYNA may help:

• However, certain foods and herbs may contain relatively high amounts of KYNA like broccoli (~2 μM), honey (~1 μM), basil (~74 μM), and thyme (~9 μM) (155, 156).

• Furthermore, the intestinal commensal Escherichia coli can produce and liberate KYNA through aspartate aminotransferase (AspAT) (153, 157, 158).

Bioavailability is high:

• KYNA is readily absorbed from the gut into the bloodstream (155). Interestingly, rats with the probiotic Bifidobacteria infantis in the gut have significantly higher KYNA levels in the blood then un-colonized control animals (159).

• Furthermore, blood from B. infantis-colonized animals exhibits a lower TNF induction after ex vivo challenge with LPS, which is a typical indication of an endotoxin tolerance.

A feedback mechanism. Keep taking your kefir, magnesium, and other pre- and probiotics too:

• There are also indications that KYNA selectively regulates the growth, and thereby the composition, of the intestinal microbiota (160). In this context, the microbial-mediated KYNA catabolism, known so far from Pseudomonas and Aerococcus (60, 61), might be relevant.

They also cautioned about taking “very high amounts of KYNA” until the studies have been done in humans. This shouldn’t be a concern, even if the source is chestnut honey, as the KYNA is still at a relatively low concentration. To my knowledge, a spoon or two a day hasn’t been associated with side effects due to KYNA!

• Interestingly, feed supplementation with very high amounts of KYNA might have a toxic/stress-inducing effect in rainbow trout (161). Hence, further studies are necessary to evaluate if a supplementation of KYNA is beneficial or detrimental to human health.

No conflicts of interest were declared.

In 2024, Alves et al. published an excellent, comprehensive literature review: “The Biology and Biochemistry of Kynurenic Acid, a Potential Nutraceutical with Multiple Biological Effects“9.

(Emphasis mine):

Abstract

Kynurenic acid (KYNA) is an antioxidant degradation product of tryptophan that has been shown to have a variety of cytoprotective, neuroprotective and neuronal signalling properties. However, mammalian transporters and receptors display micromolar binding constants; these are consistent with its typically micromolar tissue concentrations but far above its serum/plasma concentration (normally tens of nanomolar), suggesting large gaps in our knowledge of its transport and mechanisms of action, in that the main influx transporters characterized to date are equilibrative, not concentrative.

“Efflux systems function via an energy-dependent mechanism (active transport) to pump out unwanted toxic substances through specific efflux pumps. Some efflux systems are drug-specific, whereas others can accommodate multiple drugs using small multidrug resistance (SMR) transporters.^[4][5]”10

In addition, it is a substrate of a known anion efflux pump (ABCC4), whose in vivo activity is largely unknown. Exogeneous addition of L-tryptophan or L-kynurenine leads to the production of KYNA but also to that of many other co-metabolites (including some such as 3-hydroxy-L-kynurenine and quinolinic acid that may be toxic).

With the exception of chestnut honey, KYNA exists at relatively low levels in natural foodstuffs. However, its bioavailability is reasonable, and as the terminal element of an irreversible reaction of most tryptophan degradation pathways, it might be added exogenously without disturbing upstream metabolism significantly. Many examples, which we review, show that it has valuable bioactivity. Given the above, we review its potential utility as a nutraceutical, finding it significantly worthy of further study and development.

Keywords:

kynurenic acid; SLC22A6; SLC22A8; ABCC4; oxidative stress; cytoprotectant; nutraceutical

1. Introduction

Many natural products, including normal human metabolites, are of interest as candidate nutraceuticals since their deficiency, while not necessarily causing overt disease, may lead to a less-than-optimal functioning of the organism of interest [1].

Accordingly, improved functioning, and the potential for an extended and healthy lifespan, might then be realized by the addition of the nutraceutical. Research interest in such nutraceuticals, which when added to and delivered in food matrices are referred to as ‘functional foods’, is consequently considerable (e.g., [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28]).

As part of a continuing survey of nutraceuticals, where we previously focused on ergothioneine (e.g., [18,29]), we determined that kynurenic acid (KYNA) might be of nutraceutical value. Here, we bring together some of the evidence that leads us to suppose that given its somewhat limited availability in normal diets, not least as the end product of a mammalian metabolic pathway, KYNA might indeed have a nutraceutical effect when provided exogenously.

Key takeaways:

• In humans, tryptophan is one of nine essential proteinogenic amino acids that must be obtained from the diet (i.e., we do not biosynthesize it [44]), while it (and its metabolites such as KYNA) may also be produced by the gut microbiome [45,46,47,48] (and transported in a manner reflected in the gut–brain axis [49,50,51,52,53,54,55,56,57,58,59,60]) or by some other minor pathways [61].

• More than 95% of tryptophan is said to be degraded via the “L-kynurenine pathway” (KP) [62,63,64], consistent with mathematical models [65], and KYNA is one terminal part of the tryptophan degradation pathway, occurring via N-formylkynurenine and L-kynurenine (KYN) [66].

Another challenge for experimental design:

• Note, however, that not all arms of this pathway exist in all cells: they are often segregated [67,68,69,70,71,72], implying the need for single-cell analyses [73,74,75,76,77,78,79].

• … since KYNA is both seen as neuroprotective (e.g., [93,99,100]) and is essentially the terminal element and an irreversible step in this part of the kynurenine pathway, it is reasonable that provided it and its metabolites are beneficial or at least harmless, it can be added with impunity.

• Importantly, no diseases seem to be associated with the overexpression of KAT [101], the enzyme that is responsible for the synthesis of KYNA. The ability to add KYNA without affecting levels of molecules such as L-kynurenine directly is a core idea behind its potential use as a nutraceutical.

It may be useful to some gut bacteria to produce KYNA as this is immunosuppressive, increasing their survival prospects:

• Genestet et al. observed that Pseudomonas aeruginosa clinical isolates present a high transcription level of the kynaA gene (the first gene involved in the kynurenine pathway, converting L-Trp to L-kynurenine) and produce high amounts of kynurenine when in contact with human neutrophils, leading to increased bacterial survival.

They also discuss how KAT enzymes in yeast may detoxify excess tryptophan, which may be harmful to microbes. Its properties as a ROS scavenger may also benefit bacteria:

• … KYNA appeared as the best scavenger of O₂⁻ among the molecules tested [171]. In this case, an increase in L-kynurenine production by P. aeruginosa, especially when the pathway branch leading to anthranilate production is suppressed, might lead to an increase in the production of KYNA [172], which appears to be the best scavenger, although more studies are necessary to define its role over L-kynurenine in P. aeruginosa survival.

• Furthermore, KATs can play an essential role in bacterial survival and amino acid synthesis/nitrogen assimilation due to their higher affinity and efficiency for other natural substrates over L-kynurenine [168,173].

ABCC4 is a “very efficient efflux pump”:

• … they are a major cause of resistance to antibiotics (e.g., [232,253,254,255]) and to antitumor drugs (e.g., [256,257,258,259,260]). Therefore, the tissue distribution of KYNA is likely to depend strongly on the concentrations of potential ABCC4 inhibitors (some listed in Table 1) as well as the disposition of efflux transporters like ABCC4 and others of the ABCC family.

Quercetin is a potent inhibitor of ABCC4, and can therefore, in theory, increase the efficacy of many drugs such as antibiotics and chemotherapeutic agents:11

• Interestingly, among the most potent inhibitors of ABCC4 (K_i~1 μM [251]) is the flavonoid quercetin, another important nutraceutical [261,262].

If quercetin also inhibits ABCG2 then it begs the question whether the two would complement each other in your daily stack:

• ABCG2 (BCRP) is also a potential effluxer of KYNA [263,264].

BCRP is short for Breast Cancer Resistance Protein. A quick search confirms that its good news:

Difficult to model in vitro:

• Note too that kynurenic acid may also be bound to albumin [265,266], something that would be missed in standard extractive metabolomics studies.

It’s “extremely complex”:

• Overall, the system is extremely complex, benefitting strongly from the kind of ODE-based modelling that is known in this field as ‘physiologically based pharmacokinetic modelling’ [267,268], while the increasing availability of cell lines engineered to overexpress individual SLCs [269] should make answering this question of KYNA transporters much more accessible [270].

Interesting. Forget the “folic acid for all” dogma and, instead, consider dietary intake of KYNA?

• An especially striking finding [294] was that KYNA was accumulated 20-fold in cord blood relative to the maternal plasma, and its concentration was also 20-fold greater (in mice) in the fetal brain vs. maternal tissue [295] (see also [296] for external KYNA addition), strongly implying a role for both concentrative transporters and for KYNA itself in fetal development.

Tell me that this isn’t linked to Bifidobacteria-derived KYNA, too:

Quercetin has a drawback in that it has poor bioavailability:

• We note also that L-kynureine is more widely bioavailable, being a substrate of the ‘large amino acid transporter’ in C. elegans [297], a homologue of the human SLC7A5 [241,298,299,300,301,302] that transports many large, neutral amino acids, including tryptophan and L-kynurenine).

• This of course complicates analyses of the transport of KYNA if tryptophan or L-kynurenine and/or kynurenine amino-transferase(s) are also present or added (Figure 6). D-kynurenine can also be used and is metabolized via D-amino acid oxidase [303] or transamination [304].

Bioavailability of KYNA is good, including to the brain:

• Given the relative paucity and activity of (known) transporters, it is possible that KYNA is not the most bioavailable of nutraceuticals, but that oral KYNA is definitely absorbed in the mammalian gut [36,305,306,307,308], and can cross a model of the blood–brain barrier with a respectable permeability of some 3.5·10⁻⁶ cm·s⁻¹ [309], precisely that of the modal value for marketed drugs across Caco-2 cells [310].

• The levels of natural absorption can also be improved further by linking it to a transporter substrate [311,312] or using special formulations [309,313,314,315,316,317,318] (we ignore analogues that do not yield KYNA itself, since our focus is the nutraceutical activity of the genuine natural product).

• Thus, there is no reason why exogenously supplied KYNA might not be bioavailable, and the many effects reviewed here surely indicate both that it is and that can confer host benefits.

Formula milk just doesn’t compare:

• 7.1. Breast Milk

  O’Rourke et al. measured various tryptophan metabolites including KYNA in human breast milk, finding values of ca. 12 ng/mL (52 nM) at term, rising ~3.5-fold for term babies, but less so (and not significantly) in pre-term babies.

• Milart et al. [359] noted that natural human breast milk contained KYNA at much higher levels (from ca. 21 nM to ~300 nM at 6 months of breastfeeding, a ca. 14-fold increase) as lactation kicked in, levels that were well above those in a variety of commercial infant formulas.

• Rat pups exposed postnatally to KYNA also demonstrated less obesity for the same increase in bone mineral density [359] (see also [360,361]).

Some honeys are better than others as a source of KYNA:

• Honey is a notable source of KYNA. In particular, the product from sweet chestnut trees reportedly weighs in at ca. 100 mg KYNA/kg [36,307] or even more [392] (Table 3 and Figure 7).

• The source of these high levels is, in particular, the male flowers of the tree, most other parts of the edible chestnut having far lower levels [392]. Note, however, that most honeys are closer to 1 mg/kg or lower, so a 25 mg supplement (say) of KYNA would require a mighty dose of any but the most potent honey.

• An overall conclusion from Table 3 is that if KYNA is going to be given as a nutraceutical, even at low doses, its levels are likely to exceed those seen when its sole exogenous source is foodstuffs [308] (propolis, of an unstated origin, was also said to be a good source [36], although KYNA was not reported in a number of untargeted metabolomics studies [394,395,396,397], so this seems worth investigating further).

• Thus, while the precise contribution of KYNA itself is unknown (chestnut honey also contains many phenolic and other antioxidants [399]), the excellent review by Turska and colleagues [308] does provide a list of examples where this honey is thought to have provided health benefits.

• These include positive effects on glucose metabolism and neurodegeneration in obese mice [400], vs. high-fat diets in obese mice [401], acid-/alcohol-induced gastric ulceration in mice [402], on carbon tetrachloride-induced liver damage in rats [403], and in inhibiting breast cancer cell line proliferation in vitro [404].

• It has been found protective against influenza in mouse macrophages and mice in vivo [405]. Along with curcumin, it also produced a substantial increase in the longevity of heat-stressed bees [406].

KYNA should be a useful therapeutic for treating long COVID and for resisting fibrosis:

• In Table 6, we summarize some of the examples in which KYNA has proved to be neuroprotective against stresses in non-CNS tissues. Ischemia-reperfusion injury occurs when tissues subjected to hypoxia are reoxygenated, leading to the rapid formation of ROSs; although this is well known in acute circumstances, it is becoming increasingly recognized that this can also occur chronically (e.g., [413]), especially in diseases such as long COVID where fibrinaloid microclots [539,540,541] can induce hypoxia [539] and related sequelae such as reperfusion injury [413] and postural orthostatic tachycardia syndrome (POTS) [542].

• As an antioxidant, and probably via other signalling pathways, a particular feature of KYNA is its ability to lower the extent of ischemia-reperfusion injury [517].

• Another accompaniment of such diseases is fibrosis and/or amyloid deposition [543,544]. In some cases, fibrin can adopt an amyloid form, e.g., [540,545,546,547,548], though only rarely is fluorescence staining for amyloid performed [549]. Similarly, the ability of KYNA to lower fibrosis [308,550,551,552] may be relevant in this context.

Safety isn’t a concern at the doses encountered:

• Turska and colleagues provide an excellent review [308] of the possibility of enriching foodstuffs with KYNA.

High-dose tryptophan consumption also significantly increases KYNA metabolite levels. So why not simply take it in, for example, a bodybuilder’s shake? You could, and there is a case for doing this to help treat long COVID12, but it lacks the healing and antibacterial properties of the other compounds in chestnut honey, such as the phenolics and minerals:

• By and large, however, human safety studies involving dosing with substantial amounts of pure exogenous KYNA per se have largely not been performed [307], though KYNA (as chestnut honey) was given to human volunteers with no ill effects [306] while a 6 g tryptophan challenge increased KYNA levels more than 130-fold, again without seeming ill-effects, albeit some effects on cerebral blood flow in healthy controls but not in those with schizophrenia-related disorders [664] (rather implying the irrelevance of KYNA here).

• Some studies in rodents have added KYNA at massive doses (well over 100 mg/kg, getting into the millimolar range in serum/plasma), seemingly without ill effects (indeed sometimes with protective effects).

• We note that Hiratsuka and colleagues [672] gave young female participants up to 5000 mg tryptophan per day (~100 mg/kg based on the stated BMI levels) with no adverse effects; the amount of KYNA that was formed is unknown, though approximately 100 μmol/d (~19 mg) could be found in the urine [672], the value plateauing after ~7 d [673].

• Similarly, Al-Karagholi and colleagues [674] dosed volunteers with 5 mg L-kynurenine/kg (say 350 mg total) without apparent ill-effect, while Jauch et al. [675] administered L-kynurenine to rhesus monkeys at doses up to 200 mg/kg, with serum KYNA levels reaching ~25 μM within 10 min, declining to 2.8 μM after 4 h, again without apparent ill effects.

• Rentschler et al. dosed rats with L-kynurenine at 100 mg/kg, as well as a kynurenine aminotransferase inhibitor at 30 mg/kg; given that on average every marketed drug interacts with at least six known targets [676] (see later section on docking), it is hard to interpret mechanisms during such experiments in which pharmaceutical drugs are added at these kinds of concentration.

• The main point here is that while any ‘targets’ may remain unknown, there do not seem to be safety issues with these quite substantial doses.

There may be a link between KYNA and schizophrenia:

• We do also note some studies in which KYNA induced possibly undesirable effects. While far less common than those in which it has been shown to be a cytoprotectant, it is appropriate to list some of them (Table 11, see also Table 4).

• The reasons for such effects are also not really well understood, i.e., whether these are causative or they are essentially downstream responses.

• As mentioned, the biggest problem with most such studies is that they add tryptophan or L-kynurenine, which can themselves (or their other metabolites) lead to many other undocumented and important changes in host biochemistry.

• Schizophrenia seems the most common, and as noted, the evidence is at best equivocal as to whether changes in the level of KYNA are a cause or an effect or simply an accompaniment.

KYNA may not be of benefit to rheumatoid arthritis sufferers:

• 22. KYNA as a Therapeutic for Chronic Inflammatory Diseases

  Many of the diseases mentioned above share similar properties, including in particular that they are chronic and accompanied by inflammation (an interesting recent suggestion around the latter based on mitochondrial antipathy to their cellular host is worth flagging [712]).

• Thus, any nutraceuticals that might be able to tackle inflammation would be of value, and we have set out here the evidence that leads us to suppose that KYNA might be one.

• This said, there are seemingly some chronic inflammatory diseases, such as rheumatoid disease [713], that seem not to have major changes in KYNA.

Chestnut honey has been demonstrated to have beneficial effects, even with relatively low levels of KYNA:

• … our ‘index’ antioxidant nutraceutical, ergothioneine acts in part via the redox-active transcription factor Nrf2 (e.g., [741,742,743,744,745,746]). Studies in KYNA are far behind, but a tantalizing report [747] shows that chestnut honey—the foodstuff containing by far the largest amount of KYNA (Table 3)—can exert protective effects via Nrf2 on LPS-treated macrophages and indomethacin-treated gastric mucosa.

• Indeed, high levels of KYNA can induce Nrf2 synthesis [94] and prevent the induction of reactive oxygen species [95] and other changes [748] caused by quinolinic acid.

• While the aryl hydrocarbon receptor AhR is also a transcription factor, its activation can itself stimulate the activation of Nrf2 [732,733,749], and a variety of known agonists target both ArH and Nrf2 [731,750] and assist with atopic dermatitis and psoriasis (see above), so this is reasonable.

The authors also used a range of tools to model and predict which molecules KYNA is likely to interact with. It revealed an unexpected link to hypothyroidism and the use of oestrogen contraceptives:

• Remarkably, only one protein was predicted by all three tools, the thyroid hormone receptor alpha (THA, Figure 10A). This is especially interesting, as previously impaired removal of KYNA from the brain during has been observed in experimental hypothyroidism [625] (Table 8).

• Twenty-seven predicted targets for KYNA were identified by at least two of these computational compound-target prediction tools, including the aforementioned AhR (Table 12).

Concentrated in the liver, heart and kidney, PPARa helps to regulate lipid metabolism, inhibits foam cell formation (e.g. atherosclerosis and hypertension) and inhibits pro-inflammatory cytokines:

• Other nuclear receptor family members predicted in addition to THA and AhR, included the peroxisome proliferator-activated receptor alpha (PPAR-alpha) and estrogen receptor beta (ER-beta). The latter is notable in the light of a recent report of lower plasma KYNA levels in users of estrogen contraceptives [632] (Table 8).

• Not least of note is the prediction that KYNA was predicted to bind the lymphocyte specific tyrosine kinase (LCK). Critical to T cell signalling, LCK function has been shown by us to be exquisitely sensitive to dietary zinc supply [772], and this too may contribute to the immunological effects caused by KYNA.

In their conclusions, they highlight the importance of nutraceuticals as part of the bigger picture:

• Thanks to advances in scientific knowledge and in public health, human lifespan has been increasing in developed countries since the middle of the 19th century at something like 6 y per 25 y (ungendered and aggregated data for the UK at https://www.statista.com/statistics/1040159/life-expectancy-united-kingdom-all-time/, accessed on 19 August 2024), and its variation between individuals and countries has also decreased [786].

• However, the healthspan, the period in which one is free of significant ill-health, has not matched it [787]. It is widely recognized (e.g., [788,789,790]) that diet has a significant role in bringing the healthspan closer to the lifespan.

• In particular, nutraceuticals, molecules that influence health positively and that might be part of or added to a ‘healthy diet’, are seen as a contributor.

A future Substack?

• Ergothioneine (ERG), a potent and effective antioxidant, seems to be one [1,18,29,508,791,792], with experimental evidence and ongoing studies for this continuing to emerge (e.g., [320,793]), and we here make the case that KYNA is another.

• Overall, the evidence that KYNA may indeed be a worthwhile nutraceutical that might be added exogenously involves at least the following:

  • Many examples in which its exogenous addition seems to offer benefits of health or of protection against disease

  • Evidence that its concentration is relatively low in normal populations

  • Safety evidence to the effect that there do not seem to be examples in which hyperactive alleles of KAT enzymes lead to overt disease, and that exogenous KYNA cannot realistically ‘go back’ to L-kynurenine

  • Evidence that it is more or less readily bioavailable for entering plasma from the diet rather than simply being produced by compounds such as tryptophan and L-kynurenine that are more easily transported but that can lead to other, potentially toxic molecules.

• This said, not least by comparison with ergothioneine, there are considerable gaps in our knowledge of its biology.

• In the case of ergothioneine, there has been a massive upsurge in interest in the last 20 years, and as pointed out by Halliwell and Cheah [792], “a key factor was the discovery that an organic cation transporter, OCTN1, is responsible for uptake of “ergothioneine” from the gastrointestinal tract and for its distribution to tissues in the bodies of humans and other animals” (see [802,803]).

• Given the evidence for ‘missing’ transporters that we present above, a similar trajectory seems plausible for KYNA.

  ---

2.3 Other studies

Honey and osteoporosis

Honey: A Promising Therapeutic Supplement for the Prevention and Management of Osteoporosis and Breast Cancer (2023)

… Honey as a functional food with high antioxidant and anti-inflammatory properties can contribute to the prevention of various diseases. Its health benefits are mainly related to the content of polyphenols. This review aims to summarize the current knowledge from in vitro, animal, and human studies on the use of honey as a potential therapeutic agent for osteoporosis and breast cancer.

Preclinical studies have revealed a beneficial impact of honey on both bone health (microstructure, strength, oxidative stress) and breast tissue health (breast cancer cell proliferation and apoptosis, tumor growth rate, and volume).

The limited number of clinical trials, especially in osteoporosis, indicates the need for further research to evaluate the potential benefits of honey in the treatment.

Clinical studies related to breast cancer have revealed that honey is effective in increasing blood cell counts, interleukin-3 levels, and quality of life. In summary, honey may serve as a prospective therapeutic supplement for bone and breast tissue health.

More: https://www.mdpi.com/2076-3921/12/3/567

Kynurenic acid and cancer

Kynurenic acid inhibits colon cancer proliferation in vitro: effects on signaling pathways (2014)

Kynurenic acid (KYNA), a tryptophan metabolite, inhibits proliferation of several cancer cell lines including colon cancer, renal cancer and glioblastoma cells. Previous studies reported that inhibitory properties of KYNA may be related to interactions of KYNA with cell cycle regulators and signaling proteins.

… In this study, the inhibitory effect of KYNA on activation of phosphoinositide 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways in colon adenocarcinoma HT-29 cells was revealed. KYNA decreased phosphorylation of Akt, ERK 1/2 and p38 kinases in HT-29 cells.

Interestingly, the study revealed also unexpected effect of KYNA on Wnt pathway in HT-29 cells. KYNA in millimolar concentrations increased protein expression of β-catenin. However, the nuclear translocation of β-catenin in HT-29 cells exposed to KYNA was not observed.

Moreover, KYNA 1 mM increased antiproliferative properties of inhibitors of signaling pathways: wortmannin, PD98059, SB202190 and IWR-1. Taking into consideration these results, KYNA may be seen as a potential chemopreventive agent in colon cancer or supportive agent in standard cancer chemotherapy. However, the interactions between KYNA, Wnt signaling pathway and β-catenin need further studies to exclude potential effect of KYNA on colon carcinogenesis.

More: https://link.springer.com/article/10.1007/s00726-014-1790-3

HCAR3 and Kynurenic Acid in Cancer: A Promising Axis of Immunometabolic Regulation or a Scientific Mirage? (2025)

The hydroxycarboxylic acid receptor (HCAR) family belongs to G-protein-coupled receptors (GPCRs) implicated in a diverse array of physiological and pathological mechanisms. Kynurenic acid, a metabolite of the tryptophan catabolic pathway, has been proposed as a putative ligand of HCAR3. This receptor, among other HCARs, has garnered particular attention due to its exclusive expression in humans and closely related primates, and its emerging role in immunometabolic regulation.

… activation of Gi-coupled signaling cascades in immune cells by HCAR3 leads to elevated intracellular calcium levels [1,12]. HCAR3 is predominantly expressed in neutrophils, macrophages, monocytes, and basophils, supporting its pleiotropic role in systemic immune regulation. Its activation has been linked to immunomodulatory effects, including attenuation of immune responsiveness and promotion of anti-inflammatory pathways [13].

These findings indicate that HCAR3 may participate in broader immunomodulatory networks, with potential relevance to cancer development and tumor immunosurveillance. Emerging evidence implicates HCAR3 in key oncogenic processes, including cell proliferation, motility, and metabolic reprogramming of tumor cells.

Concurrently, the kynurenine pathway, the main pathway of tryptophan metabolism, is frequently dysregulated in multiple cancer types, resulting in altered production of biologically active metabolites. KYNA, an endogenous ligand of HCAR3, represents a significant metabolite of the kynurenine pathway with well-documented immunomodulatory properties.

Notably, KYNA has also been shown to modulate cancer cell metabolism, proliferation, survival, and migration, through both direct and indirect mechanisms [14,15,16,17,18].

… Direct interaction of KYNA with signaling pathways may influence the regulation of cancer cell proliferation. In vitro studies have demonstrated the antiproliferative activity of KYNA toward colon cancer, renal cancer, melanoma, and glioblastoma cells [14,15,17,18]. A comparable inhibitory effect was observed in melanoma A375 and SK-MEL-28 cells exposed to nicotinamide, another HCAR3 ligand [32]. Importantly, HCAR3 is also involved in the proliferation, differentiation, and survival of normal cells. The silencing of HCAR3 in human primary keratinocytes resulted in reduced migration and impaired metabolic activity [11].

It should be underlined that the KYNA–HCAR3 functional network may exert both direct and indirect effects on cancer cells, particularly through the modulation of immune responses. HCAR3 is highly expressed in various immune cell types, including neutrophils, monocytes, and basophils, but also in hematopoietic tissues, such as the bone marrow, and the spleen [13].

… The body of evidence and research hypotheses presented in this article suggest a potential link between the HCAR3 receptor and KYNA in the context of cancer pathophysiology. The hypothesis that KYNA may serve as a functional ligand for HCAR3, affecting cancer cell biology and modulating the immune response in the tumor microenvironment, represents a novel, interesting research direction. This concept may enhance our understanding of molecular and metabolic mechanisms of cancer progression and identify specific molecular targets for innovative therapeutic strategies.

Elucidating the potential HCAR3–KYNA axis in cancer may have significant clinical and therapeutic implications, as HCAR3 may represent a new prognostic and predictive biomarker in various cancer types, potentially facilitating personalized treatment strategies. Additionally, modulation of HCAR3 activity and/or the kynurenine pathway may represent a new therapeutic strategy in cancer treatment.

More: https://www.mdpi.com/1422-0067/26/13/6269

KYNA: “… a clear-cut dose-dependent inhibitory effect was observed in concentrations above 0.01 mM for HT-29 cells”.

HT-29 is a cell line from a female colorectal adenocarcinoma patient.

Kynurenic acid and cancer: facts and controversies (2019)

… It cannot be excluded that immune system may play an important role in biological response of cancer cells exposed to KYNA.

On the contrary, recent studies revealed an inhibitory effect of KYNA on the proliferation of human glioblastoma T98G cells (IC₅₀ = 1.3 mM) [37]. It also significantly inhibited the proliferation of human colon and renal cancer cells [24, 38]. KYNA exerted an antiproliferative activity against colon adenocarcinoma HT-29, LS-180 and Caco-2 cells with IC₅₀ of 0.9, 0.2 and 1.2 mM, respectively [24].

Importantly, it should be indicated that, a clear-cut dose-dependent inhibitory effect was observed in concentrations above 0.01 mM for HT-29 cells [24]. In vitro studies demonstrated antiproliferative properties of KYNA also against RCC Caki-2 cells (IC₅₀ = 0.04 mM) [38].

Importantly, KYNA inhibited other cellular processes in cancer cells. KYNA in millimolar concentrations significantly decreased DNA synthesis in T98G (IC₅₀ = 8.9 mM) [37], HT-29 (IC₅₀ = 4.4 mM) [63] and Caki-2 (IC₅₀ = 2.1 mM) [38] cells. It also inhibited the migration of glioblastoma (IC₅₀ = 0.6 mM) [37] and RCC [38] cells.

Although the exact molecular mechanism of the action of KYNA on cancer cells’ proliferation and motility has not been revealed, its possible interactions with receptors and several proteins involved in signal transmission and cell cycle regulation suggest the potential function of KYNA in the process of carcinogenesis (Fig. 3).

Fig. 3

More: https://link.springer.com/article/10.1007/s00018-019-03332-w

This study demonstrated that derivatives of KYNA in chestnut honey may be effective at causing apoptosis of aggressive castration-resistant prostate cancer (CRPC).

Abstract

Prostate cancer (PCa) is the most common malignancy in men and represents the second leading cause of cancer deaths in Western countries. PCa is initially androgen-dependent, however, this tumor inevitably progresses as castration-resistant prostate cancer (CRPC), which represents the most aggressive phase of the pathology.

In this work, in two CRPC cell lines (DU145 and PC3), we studied the in vitro inhibitory properties of the tryptophan-derived endogenous metabolite kynurenic acid (KYNA) and of the lactam form of 3–2′-pyrrilonidinyl-kynurenic acid (3-PKA-L), alkaloids usually present in combination in chestnut honey.

Cytotoxicity was evaluated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell colony formation assay, and Western blot analysis of the major mediator proteins involved in apoptotic processes. In all experiments, KYNA was scarcely or not active while 3-PKA-L showed anticancer activity in the high concentration range (0.01 mM – 1 mM) from 24 to 72 h.

The results obtained showed that cell death was induced by extrinsic apoptotic pathway, by cell morphological changes and reduction of cell colonies number. These novel results represent the first promising step to the accurate description of 3-PKA-L cytotoxic effect, not observed with KYNA, paving the way to the search of new anticancer agents, as well as to the better understanding of the physiopathological role of this interesting natural product.

… Recent studies that analyzed the effect of KYNA on cancer cell proliferation, demonstrated an antiproliferative activity in human glioblastoma cells (Walczak et al. 2014a, b), human colon cancer cells (Walczak et al. 2014a, b), and renal cancer cells (Walczak et al. 2012).

Besides KYNA, other structurally related quinoline alkaloids have been found in chestnut honey in a wide range of concentrations (from few mg/Kg up to hundreds of mg/Kg).

Among these derivatives, 3-PKA represents the first example of hybrid quinoline/pyrrolidine alkaloid, which involves the activated KYNA C-3 position (Fig. 1).

This is a significant finding. If you only test KYNA in the lab, you are not modelling the other relevant derivative compounds in chestnut honey.

I may need to review 3-PKA-L separately at some point.

Published data suggest this derivative as the major representative of a wider group of structurally related derivatives, which is sometimes able to even replace KYNA in the composition of chestnut honey (Beretta et al. 2009a, b; Truchado et al. 2009; Cho et al. 2015). To date, the factors underlying the origin of this metabolic shift at plant and/or honeybee level are unknown.

Keywords: Kynurenic acid, Quinoline alkaloids, Tryptophan metabolism, Prostate cancer, Apoptosis

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC8172409/

Antimicrobial properties and wound healing

Antimicrobial Evaluation of Various Honey Types against Carbapenemase-Producing Gram-Negative Clinical Isolates (2022)

The development of antibiotic resistance is a major public health issue, as infections are increasingly unresponsive to antibiotics. Emerging antimicrobial resistance has raised researchers’ interest in the development of alternative strategies using natural compounds with antibacterial activity, like honey, which has emerged as an agent to treat several infections and wound injuries.

Nevertheless, the antibacterial effect of honey was mostly evaluated against Gram-positive bacteria. Hence, the objective of our study was to evaluate the antibacterial activity, as well as the physicochemical parameters, of genuine Greek honeys against multidrug-resistant Gram-negative pathogenic bacteria.

… the antimicrobial activity of 10 different honey types was evaluated in five consecutive dilutions (75%, 50%, 25%, 12.5% and 6.25%) against the clinical isolates by the well diffusion method, as well as by the determination of the minimum inhibition concentration after the addition of catalase and protease.

Almost all the physicochemical parameters varied significantly among the different honeys. Fir and manuka honey showed the highest values in pH and H₂O₂, while the free acidity and lactonic acid levels were higher in chestnut honey.

Total phenols, total flavonoids and free radical scavenging activities were found higher in cotton, arbutus and manuka honey, and finally, manuka and oregano honeys showed higher tyrosinase inhibition activity and kojic acid levels.

… Many honeys outperformed manuka honey in their antibacterial potency. It is of interest that, for any given concentration in the well diffusion method and for any given type of honey, significant differences were not detected among the four multidrug-resistant pathogens, which explains that the damaging effect to the bacterial cells was the same regardless of the bacterial species or strain.

… Concerning, manuka honey, no inhibition was observed at concentrations 12.5% and 6.25%, while many samples of the Greek honeys showed antibacterial effects at these concentrations, such as cotton, arbutus, fir and heath at 6.25% and cotton, arbutus, chestnut, thyme, oregano, fir and heath at 12.5% concentrations (Table S1 and Figure 1a).

Almost all studied honeys exhibited antimicrobial activity at 25% and 50% concentrations, notwithstanding this effect was more pronounced at the 75% concentration, ranging from a maximum of 21.3 mm to a minimum of 13.67 mm (Table S1 and Figure 1a). Artificial honey showed a slight antibacterial effect (6.2 mm) only at a 75% concentration (Table S1).

… An analysis revealed that, in the lower concentrations, more samples outperformed the manuka honey than in higher ones (Table 4), a finding suggesting that the antibacterial factors of this honey are less effective when the honey is diluted, while the antibacterial factors of the Greek honeys are more drastic.

Manuka honey antibacterial activity has been extensively studied [43,48,49]. Thus, we enrolled manuka honey in our study as a comparative criterion for the antibacterial potency of the studied honeys.

Several Greek honeys included in our study outperformed the antibacterial effect of manuka honey (Table 4 and Table 5), regardless of their phytological origin. Arbutus, chestnut and fir honeys seemed to exhibit a more potent antimicrobial effect against our Gram-multi-resistant strains.

… The total phenols (TPC) (Table 1) were found higher in cotton (99.7 mg GAE 100 g⁻¹), fir (99.7 mg GAE 100 g⁻¹), chestnut (52.86 mg GAE 100 g⁻¹), arbutus (52.32 mg GAE 100 g⁻¹) and manuka honey (88.71 mg GAE 100 g⁻¹). Cotton and fir honey outreached the manuka honey in total phenols. Still, these two honeys (cotton and fir) showed high H₂O₂ levels superior to manuka honey. The total phenols and hydrogen peroxide showed synergistic antimicrobial activity.

… Our results showed that there are significant variations in the physical and chemical characteristics of the various types of honey, depending on their floral origin.

Furthermore, the different honeys showed important differentiations concerning their antibacterial effects, as demonstrated by the well diffusion assay, and many of them outperformed the antimicrobial potency of manuka honey.

The addition of catalase caused significant differences among the types of honeys in their antibacterial activity, assessed by the MIC method, implying that hydrogen peroxide is involved in the pathways that lead to the formation of compounds with antibacterial properties.

More: https://www.mdpi.com/2079-6382/11/3/422

Antibacterial Action Mechanisms of Honey: Physiological Effects of Avocado, Chestnut, and Polyfloral Honey upon Staphylococcus aureus and Escherichia coli (2020)

… The effects of honey samples on membrane potential, membrane integrity, and metabolic activity were assessed using different fluorochromes, in a 180 min time course assay. Time-kill experiments were also carried out under similar conditions. Exposure of S. aureus and E. coli to the distinct honey samples resulted in physiological changes related to membrane polarization and membrane integrity.

Moreover, honey induced a remarkable metabolic disruption as primary physiological effect upon S. aureus. The different honey samples induced quite similar effects on both bacteria. However, the depth of bacteria response throughout the treatment varied depending on the concentration tested and among honey varieties, probably due to compositional differences in the honey.

… The effect of honey on bacterial cell viability differed depending on the honey variety and the concentration tested. The lowest honey concentrations (20% (w/v) for AH and ChH and 10% (w/v) for PH) induced, in general, bacteriostatic effects on E. coli cells; whereas the highest concentrations (30% (w/v) for AH and ChH and 20% (w/v) for PH) produced the bacterial death. This data suggests that honey concentration is an essential factor which determine the ability of E. coli cells to resist honey induced alterations. PH exhibited the most powerful repercussion on this bacterium.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC7179468/

Chestnut honey and royal jelly in eye drops for healing corneal ulcers.

Treatment of Corneal Alkali Burn with Chestnut Honey, Royal Jelly, and Chestnut Honey-Royal Jelly Mixture (2019)

Abstract

Objectives: The alkaline burn of the cornea usually results in a decreased vision with opacification. In this study, we investigated the potential role of endemic chestnut honey (CH) and royal jelly on corneal healing after an alkaline burn.

Methods: We created an alkaline burn on the center of the corneas of four groups of Wistar rats by applying round filter paper soaked with 1 N NaOH for 30 seconds. The animals were treated with RJ, CH, RJ-CH combination and Na-Hyaluronate (Na-HA) eye drops. We performed a serial evaluation with anterior segment photography on the first, 7th, and 14th days of the experiment. Pathologic examination conducted with hematoxylin and eosin stains and immunostaining for SMA and α4β1 integrin. We evaluated the corneal healing process with a scoring system, which was estimating the degree of corneal edema, the size of the corneal ulcer, and limbal hyperemia.

Results: There was no statistically significant difference between groups on the first, 7^th, and 14^th days concerning the healing scores (p=0.88, p=0.06, p=0.80, respectively). However, there were significantly better scores in the repeated measures of CH (p=0.012) and RJ-CH (p=0.00) groups. The RJ group and Na-HA group did not show a significant difference in repeated measures (p=0.19 and p=0.10, respectively). The α4β1 integrin levels on immunostaining showed a significant difference among groups on the 14^th day (p=0.002).

Conclusion: We found better corneal healing after treatment with the RJ-CH containing eye drops concerning corneal healing sore and α4β1 integrin staining.

Keywords: Cornea; chestnut honey; corneal alkaline burn; royal jelly; smooth muscle antigen; α4β1 integrin.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC8842058/

Examination the effects of chestnut and Manuka Honey for wound healing on mice experimental model (2022)

Abstract

Objective: In this study, the aim is to examine the effects of Chestnut and Manuka honey on wound healing in order to investigate the effectiveness of cost-effective alternative to current approaches in terms of wound care and treatment.

Material and Methods: In this study, we used 30 healthy male Balbc mice weighing 18-24 g. We randomly divided the rats into three groups. A control group, a group treated with only Chestnut honey, a group treated with Manuka honey cream. After the wounds were formed in groups, the tissue samples were gathered on the seventh and fourteenth days. Then these samples were examined histologically and immunohistochemically.

Results: When the study results were evaluated, statistically significant differences were seen between histological and immune-histochemical findings in wound tissue preparations. On the seventh day, tissue samples showed re-epithelialization (P=0,002), granulation cell density (P=0,003) and angiogenesis (P= 0,003). In the fourteenth day tissue samples, we found epithelialization (P=0,001), granulation cell density (P=0,002) and angiogenesis (P=0,001). In the tissue samples in the seventh and fourteenth days between the groups, we found immino-histochemically, Ki-67 and EGF dyeing percentages as P= 0,004 and P=0,003 respectively.

Conclusion: We think chestnut honey may contribute to a shorter wound healing process.

RESULTS

When we compared the 7th and 14th day tissue samples of the control, Chestnut and Manuka honey groups in terms of reepithelialisation, statistically significant differences were found between the tissues of both days for the groups (P values in order p=0.002; p=0.001).

On the 14th day, it was observed that the reepithelialisation was completed in the chestnut and Manuka honey groups, but not in the control group. On the 7th day, it was observed that the formation of reepithelialisation in chestnut honey was quite ahead of Manuka honey group (p=0.006). It was observed that reepithelialisation was more advanced on the 7th day in chestnut and Manuka honey groups than in the control group (P values in order p=0.002; p=0.001) (Figure1)

Angiogenesis refers to the revascularisation (healing) of the wound:

… When we compared the tissue samples of the control, Chestnut and Manuka honey groups in terms of angiogenesis rates, it was observed that the tissue samples on the 14th and 7th day created statistically significant differences between the groups on both days (P value respectively p = 0.003; p = 0.001).

In the chestnut honey group, angiogenesis was found to be higher than Manuka honey and control groups on the 7th day (P values respectively p=0.005 and p=0.001 (14). The intensity of angiogenesis in the chestnut honey group on the 14th day was lower than Manuka honey and control groups (P values respectively p=0.006 and p=0.001).

Manuka honey group had a higher angiogenesis rate on the 7th day than the control group, but on the 14th day, angiogenesis was observed at higher rates in the control group than Manuka honey group.

Ki-67 staining uses an antibody to identify and quantify wound-healing, actively dividing cells:

… We stained the tissue samples on the 7th and 14th days of the standard and Western diet groups with Ki-67 and EGF antibodies separately and evaluated the results. The percentage of Ki-67 staining in the tissue samples of the Chestnut honey group on the 7th day was higher than that of Manuka honey and control groups.

… There was no statistical difference between the percentage of Ki-67 staining on the 14th day of Chestnut honey and Manuka honey groups (P>0.05). Statistically significant differences were also observed in the percentage of EGF staining between the groups on the 7th and 14th days (p=0.000). On the 7th day, the highest percentage of EGF staining was observed in Chestnut honey group. The lowest was observed in the control group.

CONCLUSION

The results obtained from this study show that honey used for therapeutic purposes in wound healing is effective in the healing process. In addition, Chestnut honey was found to be more effective than Manuka honey in wound healing. For this reason, the botanical origin of honey is extremely important in terms of productivity. Application time also affects wound healing. Based on the results from this study, new prospective studies with different honey types, animal models and dosages can be planned

More: https://medscidiscovery.com/index.php/msd/article/view/700

Impregnate a hydrogel with chestnut honey and use this to dress and heal diabetic ulcers.

Chestnut Honey Impregnated Carboxymethyl Cellulose Hydrogel for Diabetic Ulcer Healing (2017)

Abstract

Honey-based wound dressings have attracted a lot of attention from modern scientists owing to their anti-inflammatory and antibacterial effects without antibiotic resistance. Such dressings also promote moist wound healing, and have been considered natural, abundant, and cheap materials for folk marketing.

This study investigated the various behaviors and characteristics of chestnut honey-impregnated carboxymethyl cellulose sodium hydrogel paste (CH–CMC) as a therapeutic dressing, such as its moist retention, antibacterial activity for inhibiting the growth of Staphylococcus aureus and Escherichia coli, and the rate of wound healing in db/db mice. The results provide good evidence, suggesting that CH–CMC has potential as a competitive candidate for diabetic ulcer wound healing.

Keywords: chestnut honey, CMC hydrogel, γ-irradiation, wound dressing

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC6431954/

Chestnut honey has been shown to have antibacterial effects against antibiotic-resistant bacteria.

Chestnut Honey Is Effective against Mixed Biofilms at Different Stages of Maturity (2024)

Abstract

The irresponsible overuse of antibiotics has increased the occurrence of resistant bacterial strains, which represents one of the biggest patient safety risks today. Due to antibiotic resistance and biofilm formation in bacteria, it is becoming increasingly difficult to suppress the bacterial strains responsible for various chronic infections.

Honey was proven to inhibit bacterial growth and biofilm development, offering an alternative solution in the treatment of resistant infections and chronic wounds. Our studies included chestnut honey, valued for its high antibacterial activity, and the bacteria Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and S. epidermidis, known to form multi-species biofilm communities.

Minimum inhibitory concentrations (MIC) of chestnut honey were determined for each bacterial strain. Afterwards, the mixed bacterial biofilms were treated with chestnut honey at different stages of maturity (incubation times: 2, 4, 6, 12, 24 h). The extent of biofilm inhibition was measured with a crystal violet assay and demonstrated by scanning electron microscopy (SEM). As the incubation time increased and the biofilm became more mature, inhibition rates decreased gradually. The most sensitive biofilm was the combination MRSA-S. epidermidis, with a 93.5% inhibition rate after 2 h of incubation. Our results revealed that chestnut honey is suitable for suppressing the initial and moderately mature stages of mixed biofilms.

Keywords: MRSA; Pseudomonas; Staphylococcus; chronic wound.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC10967288/

Antiviral effects against herpes simplex.

Korean Chestnut Honey Suppresses HSV-1 Infection by Regulating the ROS–NLRP3 Inflammasome Pathway (2023)

Abstract

Herpes simplex virus 1 (HSV-1) is double-stranded DNA virus that belongs to the Orthoherpesviridae family. It causes serious neurological diseases of the central nervous system, such as encephalitis. The current U.S. Food and Drug Administration (FDA)-approved drugs for preventing HSV-1 infection include acyclovir (ACV) and valacyclovir; however, their long-term use causes severe side effects and often results in the emergence of drug-resistant strains.

Therefore, it is important to discover new antiviral agents that are safe and effective against HSV-1 infection. Korean chestnut honey (KCH) has various pharmacological activities, such as antioxidant, antibacterial, and anti-inflammation effects; however, antiviral effects against HSV-1 have not yet been reported.

Therefore, we determined the antiviral activity and mechanism of action of KCH after HSV-1 infection on the cellular level. KCH inhibited the HSV-1 infection of host cells through binding and virucidal steps.

KCH decreased the production of reactive oxygen species (ROS) and calcium (Ca²⁺) following HSV-1 infection and suppressed the production of inflammatory cytokines by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) activity.

Furthermore, we found that KCH inhibited the expression of the nod-like receptor protein 3 (NLRP3) inflammasome during HSV-1 infection. Taken together, the antiviral effects of KCH occur through multiple targets, including the inhibition of viral replication and the ROS-mediated NLRP3 inflammasome pathway. Our findings suggest that KCH has potential for the treatment of HSV-1 infection and related diseases.

Keywords: HSV-1, KCH, ROS, NF-кB, mitochondria, NLRP3 inflammasome

… Various honeys, including manuka, tualang, and Tilia amurensis honey, and associated compounds, such as methylglyoxal (MGO), have attracted attention as effective natural therapies because of their ability to attenuate acute inflammation by enhancing the immune response. Several studies have demonstrated antiviral effects against HIV, influenza virus, and varicella-zoster virus [34,35]. Among these, a study on Tilia amurensis honey has reported on the protective effect of influenza A virus though the activation of interferon beta signaling and the interferon inducible transmembrane protein 3 (IFITM3) pathway [35].

… In the present study, we demonstrated, for the first time, that KCH inhibits HSV replication and the ROS-mediated NLRP3 inflammasome, which is increased in HSV-1-infected cells (Figure 8). KCH inhibits viral infection by disrupting viral binding and exhibiting increased virucidal effects. In addition, we confirmed that KCH significantly reduced intracellular ROS and calcium, which were increased by viral infection.

In a previous study, we reported the anti-influenza effects of kynurenic acid (KYNA), a specific compound in chestnut honey [23]. However, we did not examine KYNA in relation to HSV-1. Therefore, we investigated the antiviral effect of KYNA on HSV-1 infection. However, KYNA did not have a significant effect. Thus, we speculate that various components in KCH other than KYNA may contribute to antiviral efficacy against HSV1. We plan to conduct further studies to analyze the various components present in KCH and study individual compounds with potential anti-HSV effects.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC10669648/

Kynurenic acid, the gut-brain axis and appetite

Intermittent or extended fasting promotes the elevation of neuroprotective KYNA, and it also favourably modulates the microbiome:

Fasting diets: what are the impacts on eating behaviors, sleep, mood, and well-being? (2024)

Introduction

The use of fasting to impact health outcomes can be traced back to the Hippocratic collection which treated seizures in a man by abstaining from food and drink (1). More recently, Guelpa and Marie (2) and later Höhn et al. (3) tested intermittent fasting as a potential intervention for epilepsy. Fasting diets (FDs) have drawn a great deal of attention among energy-restricted diet options (4) due to their associated effects on reducing serum glucose, depletion of hepatic glycogen, and a shift from glycolysis to ketogenesis. Concurrently, attention has been drawn to its various physical and mental health effects (5) such as altered anxiety behavior and cognition effects (6).

… Mounting evidence shows that FDs confer positive effects on weight control (10) and a beneficial contribution against a wide variety of health outcomes such as metabolic syndrome, type 2 diabetes mellitus (T2DM), cardiovascular diseases, and cancer (11). Moreover, several human and animal studies have reported the anti-aging effects (12) and neurological benefit of FDs. Similarly, the implementation of FDs has been shown to relieve symptoms of many disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), epilepsy (13), and stroke (14).

… Fasting diet regimens

Fasting diets have been implemented in different forms. Unlike traditional calorie restriction diets, fasting diets require a minimum of 8 h of fasting. ADF is defined as a restriction of 75% to 100% of the energy needs every other day and consuming the usual diet on the other days. The 5:2 IF which is one of the most popular regimens for FDs, involves fasting for 2 non-consecutive days and eating a habitual diet on the other 5 days of the week.

In periodic fasting, people are recommended to limit food intake for 1 or 2 days per week but are allowed to eat their regular diet for 5 or 6 days per week. TRF is characterized by eating abstinence for particular time periods of the day, generally between 8 and 12 h each day. Religious fasting regimes, such as Ramadan, in which individuals must fast from dawn to sunset for a month, are identified as other forms of fasting diets (15, 16).

… Overall, fasting benefits mental health by affecting neuroplasticity, neurogenesis, bioenergetics, and stress resistance in the nervous system. Mood comfort during fasting has been discussed as an evolutionary adaptation for survival and the search for food (111). While not fully characterized, the currently known effects of fasting on the gut-brain axis can be found in Figure 1. The neurobiology of mood improvement during periodic fasting has been discussed through several mechanisms to be discussed below (111).

… The microbiota has the potential to affect neuronal function directly or indirectly through neurotransmitters, vitamins, and microbial metabolites such as short-chain fatty acids (135). It’s difficult to understand the mechanism by which these metabolites affect brain function because intestinal-mucosal barrier and blood-brain barrier hinder a direct access to the brain (133).

It’s suggested that the microbiota may send signals to the brain by activating afferent sensory neurons of the vagus nerve via neuroimmune and neuroendocrine pathways, affecting the production of precursors, neurotransmitters, and metabolites that are associated with mood and behavior (133, 134).

The prevalent gastrointestinal ailments observed among patients with anxiety and depression disorders imply a mutual interaction between gut activities and brain function (136). Intermittent fasting has been shown to modulate the gut microbial composition and their metabolic activities towards an increased production of neurotransmitters and active metabolites. It was indicated that germ-free mice had a higher level of circulating tryptophan and low levels of hippocampal serotonin, which conversely increased when introducing tryptophan-metabolizing bacteria into the gut, along with a mitigation in anxiety-like behavior (137).

… Kyn is the main metabolite produced, with 90% of tryptophan metabolism being utilized for Kyn production (138). Kyn is subsequently metabolized into two different streams: quinolinic acid (QA) and kynurenic acid (KYNA). Traditionally, QA is viewed as neurotoxic and utilized instead for the creation of NAD⁺, whereas KYNA is viewed as neuroprotective due to its interaction with N-methyl-D-aspartate (NMDA) receptors (139).

The transition from Kyn to KYNA is governed, in part, by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). Fasting has been shown to increase the expression of PGC1α. This increase results in a higher allocation of Kyn for the production of KYNA, resulting in an increase in neuroprotection (140).

The second tryptophan metabolite, serotonin (5-HT), has two distinct types: gut-derived serotonin (GDS) and brain-derived serotonin (BDS), each with their own areas of effectiveness.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC10803520/

Fasting raises KYNA levels in the gut, and this induces appetite by inhibiting normal vagus nerve function and activating “hunger neurons” - the AgRP (Agouti-related peptide) neurons in the arcuate nucleus (ARC) of the hypothalamus.

Oral intake of KYNA does not lead to increased appetite or weight gain, unless you are already fasted. In other words, it’s a compensatory mechanism.

The gut-brain axis mechanism of normal appetite induced by kynurenic acid (2025)

Highlights

• Intestinal KYNA levels are elevated during fasting

• Oral KYNA increases food intake only in fasted mice

• KYNA activates ARC^AgRP neurons via NG-NTS circuit inputs to increase food intake

• KYNA stimulates appetite through GPR35 in intestinal vagal afferent nerve endings

Summary

Feeding is essential for both host-organism survival and gut-microbiota maintenance. Our research focuses on how kynurenic acid (KYNA), a gut-microbiota metabolite, regulates appetite during fasting. We find that fasting significantly raises KYNA levels in the intestine, which increases short-term food intake by inhibiting vagal afferent nerve in the nodose ganglion (NG) and activating AgRP neurons in arcuate nucleus (ARC^AgRP).

The orexigenic effects of KYNA are abolished by subdiaphragmatic vagotomy (sdVx), chemogenetic activation/inhibition of glutamatergic NG/ARC^AgRP neurons, inhibiting the nucleus of the solitary tract (NTS) to ARC^AgRP inputs, or knockdown of GPR35 (a KYNA receptor) in the intestinal vagal afferent nerve.

Our data support a model in which KYNA acts through the GPR35 receptor to inhibit vagal afferent signaling and subsequently activate ARC^AgRP neurons, which leads to increased food intake. These findings reveal a mechanism by which gut microbiota controls appetite during fasting, highlighting the complex relationship between microbial and host feeding behavior.

In 18-h fasting mice, oral administration of 10-mg/kg KYNA increased acute food intake after 1 h, while 100-mg/kg KYNA significantly increased acute food intake at both 1- and 3-h time points (Figure 1B). These results were replicated in three independent experiments using different cohorts of mice (Figures S1C and S1D).

Interestingly, while 1-week water supplementation of 1.25-mg/mL KYNA increased food intake (Figure 1C), it did not affect body-weight gain (Figure S1E). This discrepancy may be attributed to the previously reported stimulatory effects of chronic KYNA supplementation on energy expenditure.¹⁷

… Moreover, we found that mice had a reduced intake of the high-fat diet (HFD) in 18-h-fasting mice (Figure 1D) and the chow diet in 2-h-refed mice (Figure 1E), respectively, after oral 100-mg/kg KYNA administration.

This is consistent with Fan et al.’s finding that KYNA dose-dependent increase in chow-to-oreo intake ratio after oral administration plays a role in maintaining dietary balance.¹⁸ Additionally, a tail-vein injection of 5-mg/kg KYNA had no effect on food intake (Figure 1F). These results indicate that increasing KYNA in the gut, rather than in the blood, is important for regulating normal animal appetite.

(A) KYNA levels in intestinal contents of mice under ad libitum feeding, 18-h fasting, and 18-h fasting followed by 3-h refeed (n = 5 or 6 per group).

(B) Food intake of 18-h fasting mice after orally administering saline or 100-mg/kg KYNA (n = 6 or 7 per group).

(C) Food intake of 1 week after chronic adding KYNA to drinking water (n = 6 per group).

(D) HFD food intake of 18-h fasting mice after orally administering of saline or 100-mg/kg KYNA (n = 8 per group).

(E) Food intake of 18-h fasting followed by 2-h refed mice after orally administering saline or 100-mg/kg KYNA (n = 5 per group).

(F) Food intake of 18-h fasting mice after tail-vein injection of saline or 5-mg/kg KYNA (n = 5 or 6 per group).

Results are presented as mean ± SEM.∗p ≤ 0.05 and ∗∗p ≤ 0.01 by non-paired Student’s t test. See also Figures S1 and S2.

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… Therefore, these results demonstrate that KYNA enhances food intake by activating AgRP neurons and potentially acts through the NTS→ARC^AgRP neural circuit.

The vagus nerve endings in the intestine contain numerous receptors that sense hormones, fatty acids, and other substances.⁷ Among these, GPR35, a receptor for KYNA, is one of the most highly expressed metabolite receptors in the NG.¹⁶

Our study revealed that oral KYNA administration increased GPR35 expression in the NG. This led us to investigate whether intestinal KYNA is detected by GPR35 receptors in the gut’s vagus nerve endings. To test this hypothesis, we specifically knocked down GPR35 in the gut branch of vagal afferents by injecting retrograde shGPR35 virus into the intestine and Cre recombinase virus into the NG. This intersectional knockdown blocked KYNA’s orexigenic effect, suggesting a key mediating role of GPR35 expressed by the intestinal vagal nerve endings.

For the first time, we demonstrate that KYNA, a gut-microbiota metabolite, is detected by the GPR35 receptor in the vagal afferent nerve endings of the gut. This detection triggers a cascade where the interaction inhibits the excitability of the NG-NTS circuit, which then activates ARC^AgRP neurons to promote food intake. These findings establish KYNA’s crucial role in maintaining normal energy intake and appetite regulation.

More: https://www.sciencedirect.com/science/article/pii/S2211124725004309

Kynurenic acid and neurological conditions

Kynurenines in the Pathogenesis of Multiple Sclerosis: Therapeutic Perspectives (2020)

… 1. Introduction

Even though kynurenic acid was discovered roughly 170 years ago, it was not until the 1970s and 1980s that the kynurenine pathway (KP) sparked substantial interest among neuroscientists. This was due to the discovery that the two major products of the pathway, kynurenic acid (KYNA) and quinolinic acid (QUIN), possess significant, yet opposing effects on various neuronal cells and physiological processes [1].

The KP was found to be responsible for the overwhelming majority (>90%) of peripheric tryptophan (TRP) metabolism [2]. In the early days of kynurenine research, the belief held for a long time that the main purpose of the KP is solely the production of nicotinamide adenine dinucleotide (NAD⁺), a coenzyme already known to be a pivotal molecule in a vast amount of vital biochemical processes including, but not limited to being a key component in several redox reactions and being vital to mitochondrial function [3].

In the past decades, however, significant attention has been directed to the enzymes and metabolites of the KP, after the discovery that an alteration can be found not only in the metabolite levels but in the activity of the enzymes producing them as well in numerous disorders.

They have been implicated to play a role in neurodegenerative, psychiatric and developmental diseases, infections, tumors, autism, vascular diseases, allergies, transplant rejections, cancer immunity, immune privilege disorders and also in various autoimmune and neuroinflammatory conditions [4,5,6,7,8,9,10].

Kynurenic acid was the first member of the “kynurenine family” derived from the essential amino acid TRP. KYNA has been intensively studied in the past decades, has turned out to be a potent antagonist of excitatory ionotropic glutamate receptor on both the N-methyl-D-aspartate (NMDA) and glycineB site.

On the other hand, the other main derivate of the pathway, quinolinic acid, is a selective agonist of the aforementioned receptor. The activation of the NMDA receptors (NMDAR) results in a cationic influx (Na⁺, Ca²⁺, K⁺) to the cell; the subsequent increase in intracellular Ca²⁺ level activates several downstream signaling pathways and secondary messenger molecules, which ultimately lead to various synaptic alterations.

The superfluous activation of the NMDARs, however, causes an excessive inflow of Ca²⁺ ions, eliciting neurotoxicity and cellular damage, which can even induce neuronal cell death. Neuronal damage due to the excitotoxicity caused by excessive NMDAR activation has been linked to a number of neurodegenerative disorders including Huntington’s, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS) [11].

Additional to the confirmed excitotoxicity mediated by the overactivation of the NMDARs, an increased level of QUIN was reported in the pathogenesis of these diseases as well [4,5,6,7,8,11]. In addition to finding an elevation in QUIN levels, a decreased amount of KYNA was observed in some of these diseases. This raised the question, that not simply the increasement of QUIN is essential to the pathogenesis, but a more complex dysregulation is underlying, causing a shift of the KYNA to QUIN ratio.

… 4.5.2. Kynurenine Metabolite Changes in MS

Not only pre-clinical data are available on the kynurenines’ role in MS, but a number of studies supplied data from humans as well. In addition to the precise pathomechanism of MS being unknown yet, the exact role the KP plays in it is unclear as well. Therefore, it is not surprising that conflicting data are available regarding the various changes observed in the KP and its metabolites in MS.

There is an agreement, however, that a significant dysregulation of the KP is present in MS. Some evidence suggests, that the induction of the kynurenine pathway is mediated by pro-inflammatory cytokine-cascades, as described above [193,205,206]. Many of the previously described proinflammatory factors and cytokines modulating the KP are known to be altered in MS as well.

Therefore, it is rational to assume that changes in the KP will be present in patients with MS who show disease activity, e.g., at times of acute lesions formation/expansion, when increased inflammation in the CNS is present. It is also logical to presume that during chronic stages of the disease when little or no CNS inflammation is present only minor or no changes are expected to be seen in the KP.

Activation of the kynurenine pathway results in two very distinct and opposite events. Short-term benfits of the KP’s activation arise in the form of decreased T cell proliferation (via the previously discussed pathways and feedback loops), leading to immunosuppression, while chronic activation of the KP enzymes induces the production of neurotoxic metabolites and plays a role in preventing the innate repair mechanism of remyelination [207].

… In summary, the results of these studies suggest that the KP, most notably the activity of IDO-1, might be downregulated or unaltered in stable MS, probably contributing to disease pathogenesis, whereas its upregulation can be seen during acute inflammatory relapses, most probably reflecting an endogenous counter-regulatory reaction, which responds to anti-inflammatory therapy. A rise in downstream kynurenine metabolism additional to IDO-1 activity can be seen during an acute inflammatory exacerbation in MS.

Furthermore the imbalance between neurotoxic and neuroprotective metabolites of the kynurenine pathway favoring the neurotoxic ones might contribute to neurodegeneration in progressive MS subtypes in part via NMDA receptor-mediated excitotoxicity.

More: https://pmc.ncbi.nlm.nih.gov/articles/PMC7349747/